Compositions for delivery of plasmodium antigens and related methods

Abstract

The present disclosure provides compositions (e.g., pharmaceutical compositions) for delivery of malarial protein antigens and related technologies (e.g., components thereof and / or methods relating thereto). Among other things, the present disclosure provides polyribonucleotides encoding malarial protein antigens.

Description

COMPOSITIONS FOR DELIVERY OF PLASMODIUM ANTIGENS AND RELATED METHODS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Serial Nos. 63 / 515,067, filed July 21, 2023, 63 / 570,763, filed March 27, 2024, and 63 / 641,917, filed May 2, 2024, the entirety of each of which is incorporated by reference. BACKGROUND

[0002] Malaria is a mosquito-borne infectious disease caused by protozoan parasites of the Plasmodium genus. According to the World Health Organization, an estimated 3.4 billion people in 92 countries are at risk of being infected with the malaria parasite and developing disease. SUMMARY

[0003] The present disclosure provides technologies (e.g., compositions, methods, etc.) for delivery of Plasmodium antigens. In one aspect, provided herein is a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises one or more Plasmodium CSP polypeptide regions or portions thereof. In some embodiments, each of the one or more Plasmodium CSP polypeptide regions or portions thereof comprise 25 or more contiguous amino acids of the amino acid sequence according to SEQ ID NO: 1. In some embodiments, a “fragment” of a polypeptide is a “portion” of a polypeptide. In some embodiments, a portion is an antigenic portion.

[0004] In some embodiments, the polypeptide encoded by a provided polyribonucleotide comprises: (i) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 43), (ii) two to eighteen repeats of the amino acid sequence of NANP (SEQ ID NO: 98), and (iii) a Plasmodium CSP C-terminal region or antigenic portion thereof. In some embodiments, the polypeptide encoded by a provided polyribonucleotide comprises, in N-terminal to C-terminal order: (i) the at least two repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 43), (ii) the two to eighteen repeats of the amino acid sequence of NANP (SEQ ID NO: 98), and (iii) the Plasmodium CSP C-terminal region or antigenic portion thereof.

[0005] In some embodiments, the polypeptide encoded by a provided polyribonucleotide comprises an antigenic portion of a Plasmodium CSP C-terminal region, wherein the antigenic portion of a Plasmodium CSP C- terminal region comprises an amino acid sequence according SEQ ID NO: 239, wherein X3is N or K, X4is K, I, or R, and X5 is N or Y.

[0006] In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 240, wherein X3is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 241, wherein X3is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 243, wherein X1X2 is EK or KE, X3 is N or K, X4 is K, I, or R, and X5 is N or Y. In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 244, wherein X1X2is EK or KE, X3is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 245, wherein X1X2is EK or KE, X3is N or K, X4is K, I, or R, and X5is N or Y.

[0007] In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 246. In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 57. In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according SEQ ID NO: 60. In some embodiments, the antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 237.

[0008] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP C-terminal region or antigenic portion thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP C- terminal region.

[0009] In some embodiments, a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54.

[0010] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise an antigenic portion of a Plasmodium CSP C-terminal region. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 239-241, 243-246, 57, and 60. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 237.

[0011] In some embodiments, a polypeptide encoded by a polyribonucleotide comprises a serine immediately following the Plasmodium CSP C-terminal region or antigenic portion thereof. In some embodiments, a polypeptide comprises a serine-valine sequence immediately following the Plasmodium CSP C-terminal region or antigenic portion thereof.

[0012] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP major repeat region or antigenic portion thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP major repeat region. In some embodiments, a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence according to SEQ ID NO: 103.

[0013] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprises an antigenic portion of a Plasmodium CSP major repeat region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises at least six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following of the six repeats of the amino acid sequence of NANP.

[0014] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 101. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises eighteen repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 106.

[0015] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP minor repeat region or antigenic portion thereof. In some embodiments, a Plasmodium CSP minor repeat region or antigenic portion thereof comprises at least two repeats of the amino acid sequence of NANPNVDP.

[0016] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP minor repeat region. In some embodiments, a Plasmodium CSP minor repeat region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise an antigenic portion of a Plasmodium CSP minor repeat region.

[0017] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP N-terminal region or antigenic portion thereof. In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP N- terminal region.

[0018] In some embodiments, a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 91.

[0019] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise an antigenic portion of a Plasmodium CSP N-terminal region.

[0020] In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 85.

[0021] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP N-terminal end region.

[0022] In some embodiments, a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 88.

[0023] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP junctional region. In some embodiments, a Plasmodium CSP junctional region comprises or consists of an amino acid sequence that is at least 90% or 100% identical to the amino acid sequence of SEQ ID NO: 72.

[0024] In some embodiments, the polypeptide encoded by a polyribonucleotide comprises one or more linkers. In some embodiments, one or more linkers comprise a glycine-serine linker. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 193. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 201. In some embodiments, one or more linkers are located: (i) between two Plasmodium CSP polypeptide regions or antigenic portions thereof, (ii) between a Plasmodium CSP polypeptide region or antigenic portion thereof and a transmembrane region, (iii) between a Plasmodium CSP polypeptide region or antigenic portion thereof and a self-assembling region, and / or (iv) between a Plasmodium CSP polypeptide region or antigenic portion thereof and a multimerization region.

[0025] In some embodiments, the polypeptide encoded by a polyribonucleotide comprises a secretory signal. In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In someembodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0026] In some embodiments, a secretory signal comprises or consists of a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV-1 or HSV-2 secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 109. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 251. In some embodiments, a secretory signal is located at the N-terminus of the polypeptide.

[0027] In some embodiments, the polypeptide encoded by a polyribonucleotide comprises a transmembrane region. In some embodiments, a transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 51.

[0028] In some embodiments, a transmembrane region comprises or consists of a heterologous transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, a viral transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 174.

[0029] In some embodiments, a polypeptide does not comprise a transmembrane region.

[0030] In some embodiments, a polypeptide further comprises a Plasmodium secretory signal and a viral transmembrane region. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 51. In some embodiments, a viral transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 174. In some embodiments, a polypeptide does not comprise a transmembrane region.

[0031] In some embodiments, a polypeptide further comprises a Plasmodium secretory signal and a viral transmembrane region. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 51. In some embodiments, a viral transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region.In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 174.

[0032] In some embodiments, the polypeptide encoded by a provided polyribonucleotide comprises a multimerization region. In some embodiments, a multimerization region comprises or consists of a trimerization region. In some embodiments, a trimerization region comprises or consists of a fibritin region. In some embodiments, a fibritin region comprises or consists of an amino acid sequence according to SEQ ID NO: 191. In some embodiments, a multimerization region is located at the N-terminus of the polypeptide.

[0033] In some embodiments, the polypeptide encoded by a provided polyribonucleotide comprises a self- assembling region. In some embodiments, a self-assembling region comprises or consists of a ferritin region. In some embodiments, a ferritin region comprises or consists of an amino acid sequence according to SEQ ID NO: 248. In some embodiments, a self-assembling region is located at the N-terminus of the polypeptide.

[0034] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N- terminal end region, (iv) a Plasmodium CSP junctional region, (v) a Plasmodium CSP minor repeat region, (vi) an antigenic portion of a Plasmodium CSP major repeat region, and (vii) a Plasmodium CSP C-terminal region.

[0035] In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0036] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP.

[0037] In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 7.

[0038] In some embodiments, a polyribonucleotide comprises or consists of a ribonucleic acid sequence with at least 85% sequence identity to the ribonucleic acid sequence according to SEQ ID NO: 9. In some embodiments, a polyribonucleotide comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 9.

[0039] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP.

[0040] In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 10.

[0041] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the ribonucleic acid sequence according to SEQ ID NO: 12. In some embodiments, a polyribonucleotide comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 12.

[0042] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N- terminal end region, (iv) a Plasmodium CSP junctional region, (v) a Plasmodium CSP minor repeat region, (vi) anantigenic portion of a Plasmodium CSP major repeat region, (vii) a Plasmodium CSP C-terminal region, and (viii) a transmembrane region.

[0043] In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0044] In some embodiments, a transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 51.

[0045] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP.

[0046] In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 4. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 4.

[0047] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 6. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 6.

[0048] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N- terminal end region, (iv) a first linker, (v) a Plasmodium CSP junctional region, (vi) a Plasmodium CSP minor repeat region, (vii) an antigenic portion of a Plasmodium CSP major repeat region, (viii) a Plasmodium CSP C- terminal region, (ix) a second linker, and (x) a transmembrane region.

[0049] In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0050] In some embodiments, a transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 174.

[0051] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP.

[0052] In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 25. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 25.

[0053] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 27. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 27.

[0054] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junctional region, (v) a Plasmodium CSP minor repeat region, (vi) a Plasmodium CSP major repeat region, (vii) a linker, and (viii) a transmembrane region.

[0055] In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0056] In some embodiments, a transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 174.

[0057] In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 28. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 28.

[0058] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 30. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO 30.

[0059] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP junctional region, (iii) a Plasmodium CSP minor repeat region, (iv) an antigenic portion of a Plasmodium CSP major repeat region, (v) a first linker, (vi) an antigenic portion of a Plasmodium CSP C-terminal region, (vii) a serine-valine sequence, (viii) a second linker, and (ix) a multimerization region.

[0060] In some embodiments, a secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 109. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 251.

[0061] In some embodiments, a multimerization region comprises or consists of a trimerization region. In some embodiments, a trimerization region comprises or consists of a fibritin region. In some embodiments, a fibritin region comprises or consists of an amino acid sequence according to SEQ ID NO: 191.

[0062] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 237.

[0063] In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 31. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 31.

[0064] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 33. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 33.

[0065] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP junctional region, (iii) a Plasmodium CSP minor repeat region, (iv) an antigenic portion of a Plasmodium CSP major repeat region, (v) a first linker, (vi) an antigenic portion of a Plasmodium CSP C-terminal region, (vii) a serine-valine sequence, (viii) a second linker, and (ix) a self-assembling region.

[0066] In some embodiments, a secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 109. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 251.

[0067] In some embodiments, a polypeptide comprises a self-assembling region. In some embodiments, a self-assembling region comprises or consists of a ferritin region. In some embodiments, a ferritin region comprises or consists of an amino acid sequence according to SEQ ID NO: 248.

[0068] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP.

[0069] In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 237.

[0070] In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 34. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 34.

[0071] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 36. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 36.

[0072] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP junctional region, (iii) a Plasmodium CSP minor repeat region, (iv) an antigenic portion of a Plasmodium CSP major repeat region, (v) a first linker, (vi) an antigenic portion of a Plasmodium CSP C-terminal region, (vii) a serine-valine sequence, (viii) a second linker, and (ix) a transmembrane region.

[0073] In some embodiments, a secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal. In some embodiments, a HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 109. In some embodiments, a HSV gD secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 251.

[0074] In some embodiments, a transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 174.

[0075] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 237.

[0076] In some embodiments, a polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 40. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 40.

[0077] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 42. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 42.

[0078] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N- terminal end region, (iv) a Plasmodium CSP junctional region, (v) a Plasmodium CSP minor repeat region, (vi) a Plasmodium CSP major repeat region, (vii) a Plasmodium CSP C-terminal region variant, and (viii) a transmembrane region.

[0079] In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0080] In some embodiments, a transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region comprises or consists of an amino acid sequence according to SEQ ID NO: 51.

[0081] In some embodiments, a polypeptide comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence according to SEQ ID NO: 280. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 280.

[0082] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the nucleic acid sequence according to SEQ ID NO: 282. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 282.

[0083] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) an antigenic portion of a Plasmodium CSP N-terminal region, (iii) a first linker, (iv) a Plasmodium CSP N-terminal end region, (v) a Plasmodium CSP junctional region, (vi) a Plasmodium CSP minor repeat region, (vii) an antigenic portion of a Plasmodium CSP major repeat region, (viii) aPlasmodium CSP C-terminal region, (ix) a serine-valine sequence, (x) a second linker, and (xi) a transmembrane region.

[0084] In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0085] In some embodiments, a transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 174.

[0086] In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of according to SEQ ID NO: 85.

[0087] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP.

[0088] In some embodiments, a polypeptide comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence according to SEQ ID NO: 285. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 285.

[0089] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the nucleic acid sequence according to SEQ ID NO: 287. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 287.

[0090] One aspect provided herein relates to a polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises: (i) a secretory signal, (ii) an antigenic portion of a Plasmodium CSP N-terminal region, (iii) a first linker, (iv) a Plasmodium CSP N-terminal end region, (v) a Plasmodium CSP junctional region, (vi) a Plasmodium CSP minor repeat region, (vii) an antigenic portion of a Plasmodium CSP major repeat region, (viii) a second linker, (ix) an antigenic portion of a Plasmodium CSP C-terminal region, (x) a serine-valine sequence, (xi) a third linker, and (xii) a transmembrane region.

[0091] In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal comprises or consists of an amino acid sequence according to SEQ ID NO: 124.

[0092] In some embodiments, a transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, a HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, a HSV gD transmembrane region comprises or consists of an amino acid sequence according to SEQ ID NO: 174.

[0093] In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of according to SEQ ID NO: 85.

[0094] In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 237.

[0095] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of eighteen repeats of the amino acid sequence of NANP.

[0096] In some embodiments, a polypeptide comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence according to SEQ ID NO: 288. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 288.

[0097] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the nucleic acid sequence according to SEQ ID NO: 290. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 290.

[0098] In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises six repeats of the amino acid sequence of NANP. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region further comprises an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP.

[0099] In some embodiments, a polypeptide comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence according to SEQ ID NO: 291. In some embodiments, a polypeptide comprises or consists of an amino acid sequence according to SEQ ID NO: 291.

[0100] In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the nucleic acid sequence according to SEQ ID NO: 293. In some embodiments, a polyribonucleotide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 293.

[0101] In some embodiments, a Plasmodium is Plasmodium falciparum.

[0102] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof are one or more P. falciparum CSP polypeptide regions or antigenic portions thereof, preferably wherein Plasmodium falciparum is Plasmodium falciparum isolate 3D7.

[0103] In some embodiments, a polyribonucleotide is an isolated polyribonucleotide. In some embodiments, a polyribonucleotide is an engineered polyribonucleotide. In some embodiments, a polyribonucleotide is a codon-optimized polyribonucleotide.

[0104] In some embodiments, an RNA construct comprises in 5' to 3' order: (i) a 5' UTR that comprises or consists of a modified human alpha-globin 5'-UTR, (ii) a polyribonucleotide of any one of claims 1-192, (iii) a 3' UTR that comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA, and (iv) a polyA tail sequence.

[0105] In some embodiments, a 5' UTR comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 203. In some embodiments, a 3' UTR comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 205. In some embodiments, a polyA tail sequence is a split polyA tail sequence. In some embodiments, a split polyA tail sequence comprises or consists of a ribonucleic acid sequence according to SEQ ID NO: 207.

[0106] In some embodiments, an RNA construct further comprises a 5' cap. In some embodiments, an RNA construct further comprises a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the polyribonucleotide.

[0107] In some embodiments, a 5' cap comprises or consists of m7(3’OMeG)(5')ppp(5')(2'OMeA1)pG2, wherein A1is position +1 of the polyribonucleotide, and G2is position +2 of the polyribonucleotide.

[0108] In some embodiments, a cap proximal sequence comprises A1 and G2 of the Cap1 structure, and a sequence comprising: A3A4U5 (SEQ ID NO: 208) at positions +3, +4 and +5 respectively of the polyribonucleotide.

[0109] Another aspect provided herein relates to a composition comprising one or more polyribonucleotides according to certain embodiments described herein. In some embodiments, a composition comprises one or more RNA constructs described herein.

[0110] In some embodiments, a composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes, wherein the one or more polyribonucleotides are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

[0111] In some embodiments, a composition further comprises lipid nanoparticles, wherein the one or more polyribonucleotides are encapsulated within the lipid nanoparticles.

[0112] In some embodiments, lipid nanoparticles target liver cells. In some embodiments, lipid nanoparticles target secondary lymphoid organ cells. In some embodiments, lipid nanoparticles are cationic lipid nanoparticles.

[0113] In some embodiments, lipid nanoparticles each comprise: (a) a polymer-conjugated lipid, (b) a cationic lipid, and (c) one or more neutral lipids.

[0114] In some embodiments, a polymer-conjugated lipid comprises a PEG-conjugated lipid. In some embodiments, a polymer-conjugated lipid comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.

[0115] In some embodiments, one or more neutral lipids comprise 1,2-Distearoyl-sn-glycero-3- phosphocholine (DPSC). In some embodiments, one or more neutral lipids comprise cholesterol.

[0116] In some embodiments, a cationic lipid comprises [(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate).

[0117] In some embodiments, lipid nanoparticles have an average diameter of about 50-150 nm.

[0118] Another aspect provided herein relates to a pharmaceutical composition comprising a composition according to certain embodiments described herein and at least one pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises a cryoprotectant. In some embodiments, a pharmaceutical composition comprises an aqueous buffered solution, optionally wherein the aqueous buffered solution comprises one or more of Tris base, Tris HCl, NaCl, KCl, Na2HPO4, and KH2PO4.

[0119] Methods of administering to a subject a provided polyribonucleotide, a provided RNA construct, or a provided composition are also within the scope of the present disclosure. In some embodiments, a method comprises administering to a subject one or more doses of the pharmaceutical composition described herein.

[0120] In one aspect, a pharmaceutical composition as described herein is for use in the treatment of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject. In another aspect, a pharmaceutical composition as described herein for use in the prevention of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject.

[0121] In some embodiments, two or more doses of the pharmaceutical composition as described herein are administered to a subject. In some embodiments, three or more doses of the pharmaceutical composition asdescribed herein are administered to a subject. In some embodiments, the second of the three or more doses is administered to the subject at least 4 weeks after the first of the three or more doses is administered to the subject. In some embodiments, the third of the three or more doses is administered to the subject at least 4 weeks after the second of the three or more doses is administered to the subject.

[0122] In some embodiments, a fourth dose of the pharmaceutical composition as described herein is administered to a subject. In some embodiments, the fourth dose is administered to the subject at least one year after the third of the three or more doses is administered to the subject.

[0123] In some embodiments, a combination comprising: (i) a first pharmaceutical composition comprising a first polyribonucleotide disclosed herein, and (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second polypeptide, and the second polypeptide comprises one or more Plasmodium T-cell antigens is also provided herein.

[0124] A method comprising administering to a subject a combination according to certain embodiments described herein is also provided herein. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered on the same day. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered on different days. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject at different locations on the subject’s body.

[0125] In some embodiments, technologies described herein can be useful for treating a malaria infection. In some embodiments, technologies described herein can be useful for preventing a malaria infection. In some embodiments, a subject that is amenable to technologies described herein has or is at risk of developing a malaria infection. In some embodiments, a subject that is amenable to technologies described herein is a human.

[0126] In some embodiments, administration of a composition described herein induces an anti-malaria immune response in the subject. In some embodiments, the anti-malaria immune response in the subject comprises an adaptive immune response. In some embodiments, the anti-malaria immune response in the subject comprises a T-cell response. In some embodiments, the T-cell response is or comprises a CD4+ T cell response. In some embodiments, the T-cell response is or comprises a CD8+ T cell response. In some embodiments, the anti-malaria immune response comprises a B-cell response. In some embodiments, the anti- malaria immune response comprises the production of antibodies directed against the one or more malaria antigens.

[0127] Also within the scope of the present disclosure includes use of a pharmaceutical composition as described herein in the treatment of a malaria infection, use of a pharmaceutical composition as described herein in the prevention of a malaria infection, and use of a pharmaceutical composition as described herein in inducing an anti-malaria immune response in a subject.

[0128] Also within the scope of the present disclosure includes polypeptides encoded by polyribonucleotides according to various embodiments described herein, polypeptides encoded by RNA constructs according to various embodiments described herein, host cells comprising polyribonucleotides described herein, host cells comprising RNA constructs described herein, and host cells comprising polypeptides described herein. BRIEF DESCRIPTION OF THE DRAWING

[0129] FIG.1 includes schematics of exemplary malarial polypeptide constructs. As indicated, double circles indicate a relative position of an amino acid mutation within a construct. Additionally, empty boxeswithout a label indicate the relative location of a serine or serine valine in a construct (e.g.,93, 94, 101, 102, 103, or 105).

[0130] FIGS. 2A-2B depict in vitro expression of lipofectamine formulated RNA constructs encoding different malarial peptide constructs in HEK293T cells. FIG.2A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG. 2B shows total expression as measured by median fluorescence intensity (MFI) of expressed protein in the total HEK293T population for both transfected and non-transfected cells.

[0131] FIGS. 3A-3B depict in vitro expression of LNP-formulated RNA constructs encoding different malarial peptide constructs in HEK293T cells. FIG.3A shows transfection rate as measured by percentage of total HEK293T population that is positive for presence of expressed protein. FIG. 3B shows total expression as measured by median fluorescence intensity (MFI) of expressed protein in the total HEK293T population for both transfected and non-transfected cells.

[0132] FIGS. 4A-4B depict immunogenicity induced in mice by different formulated RNA constructs at day 21 after immunization. FIG.4A shows antibodies to an exemplary Plasmodium falciparum (Pf) CSP full length protein (“PfCSP-FL”). FIG. 4B shows antibodies to an exemplary PfCSP C-terminal domain (“C term (3D7)”). Each data point is representative of one mouse and the bar denotes mean with SEM. LDL, lower detection limit.

[0133] FIGS. 5A-5B depict immunogenicity induced in mice by different formulated RNA constructs at day 35 after immunization. FIG.5A shows antibodies to an exemplary Plasmodium falciparum (Pf) CSP full length protein (“PfCSP-FL”). FIG. 5B shows antibodies to an exemplary PfCSP C-terminal domain (“C term (3D7)”). Each data point is representative of one mouse and the bar denotes mean with SEM. LDL, lower detection limit.

[0134] FIGS. 6A-6K depict binding of antibodies generated from mice immunized with different formulated RNA constructs to various epitopes (17C, 18C, 19C, 20C, 21C, 22C, 23C, 42C, 27C, and 29C). FIG. 6A shows a visual summary of the data in FIGS. 6B-6K in the form of a heatmap. FIGS.6B-6K show bars that are representative of the area under the curve (AUC) created when plotting dilution steps versus ECL signal.

[0135] FIGS. 7A-7B depict binding specificity of antibodies generated from mice immunized with different formulated RNA constructs to CSP protein in Plasmodium falciparum sporozoite lysates. FIG. 7A shows binding between antibodies in the sera of immunized mice and CSP protein in the sporozoite (spz) lysates represented as area under the curve (AUC) created when plotting dilution steps versus luminescence signal. FIG.7B shows binding of murine anti-CSP mAb3SP2 used as a positive control.

[0136] FIGS. 8A-8C depict activation of T-cells, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG. 8A) or controls (e.g., negative control: Trp1, 2 µg / mL (FIG. 8B); positive control: concanavalin A (ConA), 2 µg / mL (FIG.8C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0137] FIGS. 9A-9C depict activation of T-cells, as assessed by secretion of IL-2. IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG. 9A) or controls (e.g., negative control: Trp1, 2 µg / mL (FIG. 9B); positive control: concanavalin A (ConA), 2 µg / mL (FIG.9C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and barsrepresent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0138] FIGS. 10A-10C depict activation of T-cells, as assessed by secretion of TNF-α. TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG.10A) or controls (e.g., negative control: Trp1, 2 µg / mL (FIG. 10B); positive control: concanavalin A (ConA), 2 µg / mL (FIG. 10C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0139] FIGS. 11A-11C depict activation of T-cells, as assessed by secretion of both IFN-γ and IL-2. IFN- γ+IL-2 secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG. 11A) or controls (e.g., negative control: Trp1, 2 µg / mL (FIG.11B); positive control: concanavalin A (ConA), 2 µg / mL (FIG. 11C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0140] FIGS. 12A-12C depict activation of T-cells, as assessed by secretion of both IFN-γ and TNF-α. IFN-γ+ TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG. 12A) or controls (e.g., negative control: Trp1, 2 µg / mL (FIG.12B); positive control: concanavalin A (ConA), 2 µg / mL (FIG. 12C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0141] FIGS. 13A-13C depict activation of T-cells, as assessed by secretion of both IL-2 and TNF-α. IL- 2+ TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG. 13A) or controls (e.g., negative control: Trp1, 2 µg / mL (FIG.13B); positive control: concanavalin A (ConA), 2 μg / mL (FIG. 13C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0142] FIGS. 14A-14C depict activation of T-cells, as assessed by secretion of IFN-γ, IL-2 and TNF-α. IFN-γ+IL-2+TNF-α secretion was assessed using isolated splenocytes (from mice immunized with different formulated RNA constructs) treated with overlapping peptide pools covering a full length CSP protein (FIG.14A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG. 14B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.14C)). Samples were measured in triplicate and negative control was measured in duplicate; each data point represents a single mouse and bars represent the group mean spot-forming units (SFU) ± SD per 5x105splenocytes. Each data point in the medium and ConA controls represents the mean of triplicates of a pool of splenocytes from all mice.

[0143] FIGS. 15A-15D depict activation of CD4 T cells only, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.15A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.15B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.15C); medium control (FIG.15D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0144] FIGS. 16A-16D depict activation of CD4 T cells only, as assessed by secretion of IL-2. IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.16A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.16B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.16C); medium control (FIG.16D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0145] FIGS. 17A-17D depict activation of CD4 T cells only, as assessed by secretion of TNF-α. TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.17A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.17B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.17C); medium control (FIG.17D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0146] FIGS. 18A-18D depict activation of CD4 T cells only, as assessed by secretion of both IFN-γ and IL-2. IFN-γ+IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.18A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.18B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.18C); medium control (FIG. 18D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0147] FIGS. 19A-19D depict activation of CD4 T cells only, as assessed by secretion of both IFN-γ and TNF-α. IFN-γ+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.19A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.19B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.19C); medium control (FIG. 19D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0148] FIGS. 20A-20D depict activation of CD4 T cells only, as assessed by secretion of both IL-2 and TNF-α. IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.20A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.20B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.20C); medium control (FIG. 20D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0149] FIGS. 21A-21D depict activation of CD4 T cells only, as assessed by secretion of IFN-γ, IL-2 and TNF-α. IFN-γ+IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD4+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.21A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.21B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.21C); medium control (FIG. 21D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD4 T cells.

[0150] FIGS. 22A-22D depict activation of CD8 T cells only, as assessed by secretion of IFN-γ. IFN-γ secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.22A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.22B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.22C); medium control (FIG.22D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0151] FIGS. 23A-23D depict activation of CD8 T cells only, as assessed by secretion of IL-2. IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.23A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.23B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.23C); medium control (FIG.23D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0152] FIGS. 24A-24D depict activation of CD8 T cells only, as assessed by secretion of TNF-α. TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.24A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.24B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.24C); medium control (FIG.24D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0153] FIGS. 25A-25D depict activation of CD8 T cells only, as assessed by secretion of both IFN-γ and IL-2. IFN-γ+IL-2 secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.25A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.25B); positive control: concanavalin A (ConA), 2 μg / mL (FIG. 25C); medium control (FIG. 25D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0154] FIGS. 26A-26D depict activation of CD8 T cells only, as assessed by secretion of both IFN-γ and TNF-α. IFN-γ+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.26A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.26B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.26C); medium control (FIG. 26D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0155] FIGS. 27A-27D depict activation of CD8 T cells only, as assessed by secretion of both IL-2 and TNF-α. IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.27A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.27B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.27C); medium control (FIG. 27D)). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0156] FIGS. 28A-28D depict activation of CD8 T cells only, as assessed by secretion of IFN-γ,IL-2 and TNF-α. IFN-γ+IL-2+TNF-α secretion was assessed by a fluorospot assay after using MACS separation to isolate CD8+ T cells (from pools of splenocytes from mice immunized with formulated RNA constructs). Cells were then incubated with overlapping peptide pools covering a full length CSP protein (FIG.28A) or controls (e.g., negative control: Trp1, 2 μg / mL (FIG.28B); positive control: concanavalin A (ConA), 2 μg / mL (FIG.28C); medium control (FIG. 28D). Pooled samples were measured in triplicate and negative control was measured in duplicate; data points and bars represent the group mean spot-forming units (SFU) ± SD per 1x105CD8 T cells.

[0157] FIGS. 29A-29F depict an assessment of antibodies generated from mice immunized with different formulated RNA constructs (as shown along the x-axis) for their ability to inhibit P. falciparum sporozoite infection of primary human hepatocytes. Percentage of inhibition of infection activity (mean with SEM) in comparison to a control (medium only, set to 0% inhibition) is shown for a 1:40 dilution (FIG. 29A), a 1:160 dilution (FIG. 29B), a 1:640 dilution (FIG. 29C), and a 1:2560 dilution (FIG.29D). Inhibition of antibodies in sera of immunized mice in ILSDA is represented as area under the curve (AUC) created when plotting dilution steps versus percentage inhibition of infection (FIG. 29E). ILSDA results for a positive control (mAb317, an antibody reported to inhibit hepatocyte infection) are also shown (FIG.29F).

[0158] FIGS. 30A-30B depict an assessment of binding and disassociation of antibodies generated from mice immunized with different formulated RNA constructs (as shown along the x-axis) and exposed to full length PfCSP, a peptide with the junction region and minor repeats (Junction + Minor repeats), or a peptide with major repeats (Major repeats). Serum samples from all mice treated with the same construct were pooled prior to analysis. Level of antibody binding to a respective binding partner is shown in resonance units (RU) (FIG. 30A). Percentage of antibody:antigen complexes still measurable after 15 minutes of dissociation (Residual Response) is calculated from initial binding (FIG.30B).

[0159] FIGS. 31A-31H depict an assessment of antibodies generated from mice immunized with different formulated RNA constructs (as shown along the x-axis) for ability to inhibit P. falciparum sporozoite traversal in HC-04 hepatoma cells. Percentage of inhibition of traversal activity (mean with SEM) in comparison to a medium control, which was set as 0% inhibition, is shown for a 1:20 dilution (FIG. 31A), a 1:40 dilution (FIG. 31B), a 1:80 dilution (FIG.31C), a 1:160 dilution (FIG.31D), a 1:320 dilution (FIG. 31E), and a 1:640 dilution (FIG. 31F). Inhibition of antibodies in sera of immunized mice in traversal assays is represented as area under the curve (AUC) created when plotting dilution steps versus % inhibition of traversal (FIG.31G). Traversal assay results for a positive control (mAb317, an antibody known to inhibit sporozoite traversal) are also shown (FIG. 31H). DEFINITIONS

[0160] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unlessotherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents each of which are hereby incorporated by reference.

[0161] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, provided compounds show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0162] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure.

[0163] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0164] Agent: As used herein, the term “agent,” may refer to a physical entity. In some embodiments, an agent may be characterized by a particular feature and / or effect. For example, as used herein, the term “therapeutic agent” refers to a physical entity has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.

[0165] Amino acid: In its broadest sense, as used herein, the term “amino acid” refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(R)– COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L- amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, suchmodification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0166] Antigen: The term “antigen”, as used herein, refers to an agent that (i) elicits an immune response (e.g., adaptive humoral immune response, cell-mediated immunity, or both); and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody.

[0167] Anti-malaria immune response: The term “anti-malaria immune response”, as used herein, refers to an immune response produced through pre-exposure to one or more Plasmodium antigens, e.g., through administration (e.g., vaccination) of the constructs as described herein directed to Plasmodium.

[0168] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0169] C-terminal domain: The term “C-terminal domain”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 273-397 of wild-type CSP sequence of Plasmodium falciparum (isolate 3D7) (SEQ ID NO:1).

[0170] C-terminal region: The term “C-terminal region”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 273-375 of wild-type CSP sequence (SEQ ID NO:1). In some embodiments, a serine follows immediately after the C-terminal region. In some embodiments, a serine and a valine follow immediately after the C-terminal region.

[0171] C-terminal region variant: The term “C-terminal region variant”, as used herein, refers to a C- terminal region that comprises one or more mutations as compared to amino acids 273-375 of wild-type CSP sequence (SEQ ID NO: 1). In some embodiments, one or more mutations are one or more substitution mutations. In some embodiments, one or more mutations comprise an indel.

[0172] Central domain: The term “central domain”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 105-272 of wild-type CSP sequence (SEQ ID NO:1).

[0173] Characteristic portion: As used herein, the term “characteristic portion”, in the broadest sense, refers to a portion of a polypeptide or region thereof whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the polypeptide or region thereof. In some embodiments, a characteristic portion of a polypeptide or region thereof is a portion that is found in the polypeptide or region thereof and in related polypeptide or region thereof that share the particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristicportion shares at least one functional characteristic with the intact polypeptide or region thereof. For example, in some embodiments, a “characteristic portion” of a polypeptide or region thereof is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of the polypeptide or region thereof. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a polypeptide or region thereof (e.g., CSP, its N terminal domain, its major repeat region etc.) is one that, in addition to the sequence and / or structural identity specified above, shares at least one functional characteristic with the relevant intact polypeptide or region thereof. In some embodiments, a characteristic portion may be biologically active. In some embodiments, a fragment as described herein can be a portion. Accordingly, in some embodiments, a characteristic fragment can be a “characteristic portion.”

[0174] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents (e.g., two or more antibody agents)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0175] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0176] Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chainbut rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS- BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0177] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” (or “therapeutic regimen”) may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.

[0178] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0179] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0180] Heterologous: As used herein, the term “heterologous”, with respect to secretory signal or transmembrane region, refers to a secretory signal or transmembrane region from a virus or an organism other than Plasmodium.

[0181] Homology: As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0182] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0183] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be “increased” relative to that obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Thoseskilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0184] In order: As used herein with reference to a polynucleotide or polyribonucleotide, “in order” refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with reference to a polypeptide, “in order” refers to the order of features moving from the N-terminal-most of the features to the C-terminal-most of the features along the polypeptide. “In order” does not mean that no additional features can be present among the listed features. For example, if Features A, B, and C of a polynucleotide are described herein as being “in order, Feature A, Feature B, and Feature C,” this description does not exclude, e.g., Feature D being located between Features A and B.

[0185] Isolated: The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0186] Junction region: The term “junction region”, as used herein, refers to a region of a CSP polypeptide that corresponds to amino acids 93-104 of wild-type CSP sequence (SEQ ID NO:1).

[0187] Linker: As used herein, the term “linker” refers to a portion of a polypeptide that connects different regions, portions, or antigens to one another.

[0188] Lipid: As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0189] Major repeat region: As used herein, the term “major repeat region” refers to a region of a CSP polypeptide that corresponds to amino acids 129-272 of wild-type CSP sequence (SEQ ID NO:1) and contains 35 repeats of the amino acid sequence NANP (SEQ ID NO: 147). The 35 repeats of the amino acid sequence NANP (SEQ ID NO: 98) are separated into two contiguous stretches, the first stretch containing 17 repeats of the amino acid sequence NANP (SEQ ID NO: 98) and second stretch containing 18 repeats of the amino acid sequence NANP (SEQ ID NO: 98) which flank an amino acid sequence of NVDP (SEQ ID NO: 97). A portion (e.g., antigenic portion) of the major repeat region contains at least the amino acid sequence NPNA (SEQ ID NO: 96). In some embodiments a portion (e.g., antigenic portion) of the major repeat region contains at least the amino acid sequences NANPNA (SEQ ID NO: 100) and NPNANP (SEQ ID NO: 99). In some embodiments, a portion (e.g., antigenic portion) of the major repeat region comprises at least the amino acid sequence NPNA. As used herein, “repeat” in reference to sequence A refers to sequence A being present once, and “one or more repeats” of sequence A refers to sequence A being present one or more times.

[0190] Merozoite stage specific Plasmodium antigen: As used herein, the term “merozoite stage specific Plasmodium antigen” refers to an antigen that is expressed during the merozoite stage of the Plasmodium life cycle.

[0191] Minor repeat region: As used herein, the term “minor repeat region” refers to a region of a CSP polypeptide that corresponds to amino acids 105-128 of wild-type CSP sequence (SEQ ID NO:1) and contains 3 repeats of the amino acid sequence NANPNVDP (SEQ ID NO: 43). A minor repeat region does not contain the amino acid sequence NPNA (SEQ ID NO: 96) and does not contain the amino acid sequence NANPNA (SEQ ID NO: 100) or NPNANP (SEQ ID NO: 99). As used herein, “repeat” in reference to sequence A refers to sequence A being present once, and three repeats of sequence A refers to sequence A being present three times.

[0192] Multimerization region: As used herein, the term “multimerization region” refers to a region that directs assembly of multimers into a complex, where each multimer comprises a polypeptide associated with the multimerization region.

[0193] N-terminal domain: As used herein, the term “N-terminal domain” refers to a region of a CSP polypeptide that corresponds to amino acids 19-104 of wild-type CSP sequence (SEQ ID NO:1).

[0194] N-terminal start region: As used herein, the term “N-terminal start region” refers to a region of a CSP polypeptide that corresponds to amino acids 19-31 of wild-type CSP sequence (SEQ ID NO:1).

[0195] N-terminal end region: As used herein, the term “N-terminal end region” refers to a region of a CSP polypeptide that corresponds to amino acids 81-92 of wild-type CSP sequence (SEQ ID NO:1).

[0196] N-terminal region: As used herein, the term “N-terminal region” refers to a region of a CSP polypeptide that corresponds to amino acids 19-80 of wild-type CSP sequence (SEQ ID NO:1).

[0197] RNA lipid nanoparticle: As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0198] Neutralization: As used herein, the term “neutralization” refers to an event in which binding agents such as antibodies bind to a biological active site of a parasite such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminates or significantly reduces ability of a parasite to infect cells.

[0199] Nucleic acid / Polynucleotide: As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In someembodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N- phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non- natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0200] Pharmaceutically effective amount: The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease (e.g., malaria), a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., malaria). In some embodiments, such inhibition may comprise slowing down the progress of a disease (e.g., malaria) and / or interrupting or reversing the progress of the disease (e.g., malaria). In some embodiments, a desired reaction in a treatment of a disease (e.g., malaria) may be or comprise delay or prevention of the onset of a disease (e.g., malaria) or a condition (e.g., a malaria associated condition). An effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on disease (e.g., malaria) or a condition (e.g., a malaria associated condition) to be treated, the severity of such a disease (e.g., malaria) or a condition (e.g., a malaria associated condition), individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of a composition (e.g., a pharmaceutical composition) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0201] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a polypeptide is a malarial polypeptide construct described herein. A malarial polypeptide construct is a polypeptide that includes one or more malarial proteins, or one or more portions thereof (e.g., antigenic portion). In some embodiments, a malarial polypeptide construct described herein includes at least one region of Plasmodium CSP or a portion thereof (e.g., antigenic portion). In some embodiments, a malarial polypeptide construct additionally includes one or more additional amino acid sequences, such as a secretory signal (e.g., a heterologous secretory signal), a transmembrane region (e.g., a heterologous transmembrane region), a helper antigen, a multimerization region, and / or a linker, as described herein.

[0202] Prevent: As used herein, the term “prevent” or “prevention” when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time. In some embodiments, prevention refers to reducing the risk of developing clinical malaria.

[0203] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0204] Ribonucleic acid (RNA) or Polyribonucleotide: As used herein, the term “ribonucleic acid,” “RNA,” or “polyribonucleotide” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is a mRNA. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 3' end a poly(A) region. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods). In some embodiments, a polyribonucleotide encodes a polypeptide, which is preferably is a malarial polypeptide construct.

[0205] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0206] Secretory signal: As used herein, the term “secretory signal” refers to an amino acid sequence motif that targets associated polypeptides for translocation to a secretory pathway.

[0207] Subject: As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In preferred embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., malaria and / or a malaria- associated condition). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0208] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0209] Susceptible to: An individual who is “susceptible to” a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) is one who has a higher risk of developing the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition (e.g., malaria and / or a malaria-associated condition) may not have been diagnosed with the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) may exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) may not exhibit symptoms of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) will develop the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition) will not develop the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition).

[0210] Therapy: The term “therapy” refers to an administration or delivery of an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, a therapeutic agent or therapy is a medical intervention that can be performed to alleviate,relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0211] Transmembrane region: As used herein, the term “transmembrane region” refers to a region of a polypeptide that spans a biological membrane, such as the plasma membrane of a cell.

[0212] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition). In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition (e.g., malaria and / or a malaria-associated condition).

[0213] Variant: As used herein, the term “variant” refers to a molecule that shows significant structural (e.g., primary or secondary) identity with a reference molecule but differs structurally from the reference molecule. For example, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS I. Malaria

[0214] Malaria is a mosquito-borne infectious disease caused by single-celled eukaryotic Plasmodium parasites that are transmitted by the bite of Anopheles spp. mosquitoes (Phillips, M., et al. Malaria. Nat Rev Dis Primers 3, 17050, 2017, which is incorporated herein by reference in its entirety). Mosquitoes that transmit malaria must have been infected through a previous blood meal taken from an infected subject (e.g., a human). When a mosquito bites an infected subject a small amount of blood is taken in containing Malaria parasites. The infected mosquito can then subsequently bite a non-infected subject, infecting the subject.

[0215] Malaria remains one of the most serious infectious diseases, causing approximately 200 million clinical cases and 500,000-600,000 deaths annually. Although significant effort has been invested in developing therapeutic treatments for malaria, many malaria parasites have developed resistance to available therapeutics. According to Malaria Eradication Research Agenda Initiative, malaria eradication will only be achievable through effective vaccination.

[0216] In 2015, the European Medicines Agency gave a positive review to a malaria vaccine candidate known as “RTS,S”, a milestone in malaria vaccine development. In 2019, the World Health Organization launched pilot programs that provide RTS,S to children at least 5 months of age in parts of three sub-Saharan African countries. RTS,S / AS01 is an adjuvanted protein subunit vaccine that consists of a portion of the major repeat region and the C-terminus of CSP from Plasmodium falciparum fused to the Hepatitis B surface antigen (HBsAg). The vaccine is a mix of this PfCSP-HBsAg compound with HBsAg that forms virus-like particles (RTS,S / AS01; Mosquirix™). RTS,S is administered according to a regimen that requires four doses: an initial 3-dose schedule given at least 1 month apart, and a 4th dose 15-18 months after dose 3 (see, for example, Vandoolaeghe &Schuerman Expert Rev Vaccines. 15:1481, 2016; PATH_MVI_RTSS_Fact Sheet_042019, each of which is incorporated herein by reference in its entirety). Reports indicate that RTS,S protects approximately 30% to 50% of children from clinical disease over 18 months. RTS,S has been reported to induce protective antibody and CD4+ T-cell responses, but only negligible CD8+ T cell responses (see, for example, Moris et al. Hum Vaccin Immunother 14:17, 2018, which is incorporated herein by reference in its entirety). Phase III studies of RTS,S delivered as a three-dose series with a booster after 1 yr (year) showed moderate vaccine efficacy in children aged 5 to 17 months preventing 36% of clinical malaria cases over the full study period with a median follow-up of 4 yrs, with a range of 20% in high to 66% in low transmission settings. Furthermore, published literature suggests that protection wanes over time including reports of potential negative efficacy after 5 yrs in children with high malaria exposure (Olotu et al. 2016, N. Engl. J. Med. 374:2519-29) which is incorporated herein by reference in its entirety). Thus, an effective malaria vaccine remains an unmet medical need of critical importance for global health. A. Lifecycle

[0217] During a blood meal, infected mosquitos inject, along with their anticoagulating saliva, sporozoites known as the liver stage of Plasmodium spp. Sporozoites journey through the skin to the lymphatics and into hepatocytes of the liver. This journey happens very quickly; it can be complete within only a few minutes (Sinnis et al., Parasitol Int. 2007 Sep;56(3):171-8, which is incorporated herein by reference in its entirety). This is a time known to be a bottleneck of Malaria infection most favorable for therapeutic intervention, as only a small number (thought to be a few hundred at maximum) of sporozoites are injected by the mosquito, with only fraction of that number establishing infection in the liver and developing into mature live-stage parasites (Flores- Garcia et al., mBio. 2018 Nov 20;9(6):e02194-18, which is incorporated herein by reference in its entirety). Thus, a subject whose immune system is primed to clear sporozoites before they enter hepatocytes can efficiently clear an infection.

[0218] One particular challenge associated with clearing a malarial infection during this bottle neck is that the most abundant and immunogenic protein on the sporozoite surface, the circumsporozoite protein (CSP), is only exposed to the immune system in small quantities and for short duration of time due to the variably low inoculum from the mosquito and the kinetics of hepatocyte infection after inoculation. After liver infection is established, the parasite differentiates into a stage which no longer expresses CSP and instead has a different mosaic of surface antigens. Furthermore, due to the density and close proximity of neighboring CSPs on the surface of the parasite coupled with the bi-valency of antibodies, binding of antibodies to CSP can produce a phenomenon referred to as CSP precipitation reaction, whereby antibodies can crosslink neighboring CSP and cause them to precipitate and shed from the parasite surface, leaving a trail of precipitated antibody bound CSP that the parasite can replace through its normal CSP translocation process (Livingstone et al., Sci Rep 11, 5318 (2021); Steward et al., J Protozool. 1991 Jul-Aug; 38(4):411-21, each of which is incorporated herein by reference in its entirety).

[0219] When moving from an inoculation site in the skin to the liver, sporozoites traverse host cells (Mota et al., Science 2001 Jan 5;291(5501):141-4). Sporozoites traverse different types of host cells at the dermis, including fibroblasts and phagocytes (Amino et al., Cell Host Microbe. 2008 Feb 14;3(2):88-96, which is incorporated herein by reference in its entirety), and the liver sinusoidal barrier, containing liver endothelial cells and Kupffer cells (Frevert et al., PLoS Biol 3(6): e192.2005, which is incorporated herein by reference in its entirety) and sinusoidal endothelial cells (Tavares et al., J Exp Med 2013 May 6;210(5):905-15, which is incorporated herein by reference in its entirety), in order to gain access to hepatocytes. Sporozoites preferentiallytraverse cells with low-sulfated heparin sulfate proteoglycans (HSPGs) but preferentially invade cells with high- sulfated HSPGs (Coppi et al., Cell Host & Microbe 2, 316–327, November 2007, which is incorporated herein by reference in its entirety).

[0220] Cell traversal was first observed as non-phagocytic entry of P. berghei sporozoites into macrophages followed by “escape” from these cells (Vanderberg et al., J. Euk. Microbiol.37:528-536, 1990, which is incorporated herein by reference in its entirety). The biochemical, biophysical, and stepwise processes of traversal are still being explored. However, it has been suggested by electron microscopy that host cell rupture occurs upon entry and exit from the host cell (Mota et al., 2001; Tavares et al., 2013, each of which is incorporated herein by reference in its entirety). It has also been shown that P. yoelii sporozoites can enter hepatocytes via a transient vacuole and that host membrane rupture occurs upon cell exit rather than cell entry (Risco-Castillo et al., Cell Host Microbe 2015 Nov 11;18(5):593-603, which is incorporated herein by reference in its entirety).

[0221] Sporozoites also traverse hepatocytes before establishing a productive hepatocyte infection (Mota et al., 2001, which is incorporated herein by reference in its entirety). Several possibilities emerged as to why this occurs. The first hypothesis suggested that migration through hepatocytes primes parasites for invasion by activating apical exocytosis (Mota et al., Nat Med 2002 Nov;8(11):1318-22, which is incorporated herein by reference in its entirety). The second theory suggested that traversal releases hepatocyte growth factor (HGF), making neighboring hepatocytes more susceptible to infection (Carrolo et al., Nat Med. 2003 Nov;9(11):1363-9, which is incorporated herein by reference in its entirety). Lastly, other studies suggest that it takes some time for sporozoites to switch off the machinery for traversal and activate invasion machinery (Amino et al., 2008, Coppi et al., 2007, each of which is incorporated herein by reference in its entirety), and that traversal primarily functions to penetrate cell barriers and avoid phagocytosis en route to the liver (Amino et al., 2008, Coppi et al., 2007, Tavares et al., 2013, each of which is incorporated herein by reference in its entirety).

[0222] Although it has been shown that sporozoites traverse human cells (Behet et al., Malar J 2014 Apr 5;13:136; Cha et al., J Exp Med 2015 Aug 24;212(9):1391-403; Dumoulin et al., PLoS One 2015 Jun 12;10(6):e0129623; van Schaijk et al., PLoS ONE, 3 (10). e35492008, each of which is incorporated herein by reference in its entirety), the molecular basis for the traversal process is largely unstudied. Antibodies against circumsporozoite protein (CSP) impair traversal (Dumoulin et al., 2015, which is incorporated herein by reference in its entirety), but this is likely due to inhibition of motility rather than a direct effect (Cha et al., J Exp Med 2016 Sep 19;213(10):2099-112, which is incorporated herein by reference in its entirety). Furthermore, antibodies induced by chloroquine prophylaxis with sporozoites interfere with cell traversal, and these may also target CSP (Behet et al., 2014, which is incorporated herein by reference in its entirety). Recently it was shown that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the parasite surface interacts with CD68 on Kupffer cells during traversal (Cha et al., 2015, Cha et al., 2016, each of which is incorporated herein by reference in its entirety).

[0223] In rodent malaria parasites such as P. berghei, two sporozoite microneme proteins have been identified that appear to be essential for cell traversal (sporozoite microneme protein essential for cell traversal (SPECT1; Ishino et al., PLoS Biol., 2 (2004), pp. 77-84, which is incorporated herein by reference in its entiret)] and SPECT2 (Ishino et al., Cell. Microbiol., 7 (2005), pp. 199-208, which is incorporated herein by reference in its entirety), also called perforin-like protein 1 (PLP1) (Kaiser et al., Mol. Biochem. Parasitol., 133 (2004), pp.15-26, which is incorporated herein by reference in its entirety). Even though genetic disruption of SPECT1 or SPECT2 rendered sporozoites unable to traverse murine cells, they still invaded hepatocytes in vitro (Ishino et al., 2004,Ishino et al., 2005, each of which is incorporated herein by reference in its entirety). When injected into rodents, sporozoites lacking SPECT1 or SPECT2 were impaired for liver infection, but a small number of sporozoites could still establish liver infection that resulted in subsequent patency. However, depletion of Kupffer cells allowed mutants to establish liver infection at levels comparable with wild-type parasites (Ishino et al., 2004, Ishino et al., 2005, each of which is incorporated herein by reference in its entirety). This data suggests that traversal by rodent-infecting sporozoites is important for navigating through the sinusoidal layer, but not for hepatocyte invasion, malarial exoerythrocytic forms development, or growth within erythrocytes (Ishino et al., 2004, Ishino et al., 2005, each of which is incorporated herein by reference in its entirety).

[0224] The ortholog of SPECT2 in P. yoelii, PLP1, has been shown to play a role in cell traversal. Although this protein is not required for hepatocyte entry, it plays a role in egress from transient vacuoles during traversal (Risco-Castillo et al., 2015, each of which is incorporated herein by reference in its entirety). Thus, sporozoites that infect rodents can traverse host cells by generating a vacuole at the entry step and use a perforin-like protein (e.g., SPECT2 / PLP1) to escape from this compartment and / or a host cell, during cell exit.

[0225] Once sporozoites have invaded liver cells, they differentiate into merozoites, a replicative form of the parasite capable of lysing hepatocytes after multiple rounds of replication. Within a few days, a few hundred sporozoites can become hundreds of thousands of merozoites. When infected liver cells rupture, they release the merozoites into the bloodstream, where they invade red blood cells and begin the asexual reproductive stage, which is the symptomatic stage of the disease. Within a small number of days, millions of merozoites can be present in blood.

[0226] Malaria symptoms typically develop 4-8 days after initial red blood cell invasion. Replication cycle of merozoites within the red blood cells continues for 36-72 hours, until hemolysis, releasing the merozoites for another round of red blood cell infection. Thus, in synchronous infections (infections that originate from a single infectious bite), fever occurs every 36–72 hours, when infected red blood cells lyse and release endotoxins en masse.

[0227] Plasmodium spp. parasites gain entry into red blood cells through specific ligand–receptor interactions mediated by proteins on the surface of the parasite that interact with receptors on the host erythrocyte (mature red blood cell) or reticulocyte (immature red blood cell), whereas P. falciparum can invade and replicate in erythrocytes and reticulocytes, P. vivax and other species predominantly invade reticulocytes, which are less abundant than erythrocytes. Most of the erythrocyte-binding proteins or reticulocyte-binding proteins that have been associated with invasion are redundant or are expressed as a family of variant forms; however, for P. falciparum, two essential red blood cell receptors (basigin and complement decay-accelerating factor (also known as CD55)) have been identified.

[0228] Plasmodium vivax and Plasmodium ovale can also enter a dormant state in the liver, the hypnozoite.

[0229] Merozoites released from red blood cells can invade other red blood cells and continue to replicate, or in some cases, they differentiate into male or female gametocytes. Gametocytes concentrate in skin capillaries and are then taken up by the mosquito vector in another blood meal. In the gut of the mosquito, each male gametocyte produces eight microgametes after three rounds of mitosis; the female gametocyte matures into a macrogamete. Male microgametes are motile forms with flagellae and seek the female macrogamete. The male and female gametocytes fuse, forming a diploid zygote, which elongates into an ookinete; this motile form secretes a chitinase in order to enter the peritrophic membrane and traverse the midgut epithelium to the basal lateral side of the midgut, establishing itself in the basal lamina as an oocyst. Oocysts mature over 14-15 days,undergoing cycles of replication to form sporozoites that are ultimately liberated into the hemocoel, an environment rich in sugars and substrates beneficial to the parasite’s survival. Thousands of sporozoites can form from a single oocyst and become randomly distributed throughout the hemocoel. These sporozoites are motile and rapidly destroy the hemolymph, with only approximately 20% successfully invading the salivary gland. Following invasion of the salivary gland, sporozoites are re-programmed via an unknown mechanism to prepare for liver invasion. Evidence of this reprogramming has been demonstrated by the inability of midgut sporozoites (directly from oocysts) to invade hepatocytes, and also by the fact that sporozoites which have successfully invaded a salivary gland are unable to do re-invade another salivary gland if presented one. Salivary gland sporozoites alter mosquito behavior and salivary gland function, as less saliva is produced resulting in an increase in mosquito probing behavior, increasing the chances of transmission to a human host via a mosquito bite.

[0230] Some drugs that prevent Plasmodium spp. invasion or proliferation in the liver have prophylactic activity, drugs that block the red blood cell stage are required for the treatment of the symptomatic phase of the disease, and compounds that inhibit the formation of gametocytes or their development in the mosquito (including drugs that kill mosquitoes feeding on blood) are transmission-blocking agents (Phillips, et al. Malaria. Nat Rev Dis Primers 3, 17050 (2017), which is incorporated herein by reference in its entirety). B. Genome

[0231] Since completion of the first sequence of P. falciparum 3D7 genome in 2002, genomic research on malaria parasites has rapidly advanced. Except for a short diploid phase after fertilization in the mosquito midgut, Plasmodium parasites are haploid throughout their life cycle. The genomes of different species range from 20 to 35 megabases, contain 14 chromosomes, a circular plastid genome of approximately 35 kilobases, and multiple copies of a 6 kilobase mitochondrial DNA. Comparison of genomes from different species showed that homologous genes are often found in synthetic blocks arranged in different orders among different chromosomes.

[0232] The adenine-thymine (AT) content of Plasmodium spp. can also be very different, e.g., ∼80% AT in P. falciparum, P. reichenowi, and P. gallinaceum; ∼75% AT in rodent malaria parasites; and ∼60% AT in P. vivax, P. knowlesi, and P. cynomolgi. AT content is often higher in introns and intergenic noncoding regions than in protein-coding exons, with an average of 80.6% AT for the whole P. falciparum genome versus 86.5% for noncoding sequences. The high AT content of P. falciparum reflects large numbers of low-complexity regions, simple sequence repeats, and microsatellites, as well as a highly skewed codon usage bias. Polymorphisms of AT-rich repeats provide abundant markers for linkage mapping of drug resistance genes and for tracing the evolution and structure of parasite populations.

[0233] Malaria parasite genomes carry multigene families that serve important roles in parasite interactions with their hosts, including, for example, antigenic variation, signaling, protein trafficking, and adhesion. Among the gene families, genes encoding P. falciparum erythrocyte membrane protein 1 (PfEMP1) have been studied most extensively. Each individual P. falciparum parasite carries a unique set of 50 to 150 copies of the var gene in its genome, where switches of gene expression can produce antigenic variation. PfEMP1 plays an important role in the pathogenesis of clinical developments such as in cerebral and placental malaria, in which it mediates the cytoadherence of infected red blood cells (iRBCs; infected erythrocytes) in the deep tissues. Different PfEMP1 molecules bind to various host molecules, including α2-macroglobulin, CD36, chondroitin sulfate A (CSA), complement 1q, CR1, E-selectins and P-selectins, endothelial protein C receptor (EPCR), heparan sulfate, ICAM1, IgM, IgG, PECAM1, thrombospondin (TSP), and VCAM1. Such binding leads to activation of various host inflammatory responses. Hemoglobinopathies, including the hemoglobin C andhemoglobin S trait conditions, interfere with PfEMP1 display in knob structures of the iRBCs. This poor display of PfEMP1 on the host cell surface offers protection against malaria by reducing the cytoadherence and activation of inflammatory processes that promote the development of severe disease.

[0234] Members of the large Plasmodium interspersed repeat (pir) multigene family are named differently by parasite species, for example, yir in P. yoelii, bir in P. berghei, vir in P. vivax. Several P. falciparum gene families (stevor, rif, and PfMC-2TM) are classified with pir by their similar gene structures, which characteristically include a short first exon, a long second exon, and a third exon encoding a transmembrane domain. In a recent study, the pir genes from P. chabaudi (cir) were shown to be expressed in different cellular locations, within and on the surface of iRBCs, and in merozoites. Malaria parasites devote large portions of their genomes to gene families that ensure evasion of host immune defenses and protection of molecular processes essential to infection. These families emphasize the importance of research on their roles in parasite-host interactions and virulence, despite the difficulties inherent to their investigation.

[0235] An additional, exemplary polymorphic gene family comprises a group of 14 genes encoding proteins with six cysteines (6-Cys). These proteins often localize on the parasite surface interacting with host proteins and are expressed at different parasite developmental stages.6-Cys proteins also demonstrate diverse functions and have been shown to play roles in, for example, parasite fertilization, mating interactions, evasion of immune responses, and invasion of hepatocytes. The proteins expressed in asexual stages are generally polymorphic and / or under selection, suggesting that they could be targets of the host immune response; however, their functions in parasite development remain largely unknown.

[0236] Plasmodium genomes can be highly polymorphic. Early studies demonstrated polymorphisms involving tens to hundreds of kilobases and that the chromosome structure in P. falciparum is largely conserved in central regions but extensively polymorphic is both length and sequence near the telomeres. Much of the subtelomeric variation was explained by recombination within blocks of repetitive sequences and families of genes.

[0237] The frequency of simple sequence repeats (microsatellites) in P. falciparum is estimated to be approximately one polymorphic microsatellite per kb DNA. Without wishing to be bound by any one theory, this high rate may reflect the AT-rich nature of the genome. Microsatellites seem to be less frequent in other Plasmodium species that have genomes with lower AT contents. In addition to the highly polymorphic and repetitive structure of Plasmodium genomes, there are also large numbers of Single Nucleotide Polymorphisms (SNPs) and Copy Number Variations (CNVs) (Su et al., Plasmodium Genomics and Genetics: New Insights into Malaria Pathogenesis, Drug Resistance, Epidemiology, and Evolution. Clin Microbiol Rev.2019 Jul 31;32(4), which is incorporated herein by reference in its entirety). C. Malarial Proteins

[0238] Plasmodium parasites are known to express various proteins at different stages of their lifecycles. Exemplary malarial proteins are described below, and exemplary amino acid sequences are provided in Table 2.

[0239] Circumsporozoite protein (CSP) is a multifunctional protein that is involved in Plasmodium life cycle, as it is required for the formation of sporozoites in the mosquito midgut, the release of sporozoites from the oocyst, invasion of salivary glands, attachment of sporozoites to hepatocytes in the liver, and sporozoite invasion of hepatocytes (see, e.g., Zhao et al. (2016) PLoS ONE 11(8): e0161607, which is incorporated herein by reference in its entirety). CSP is present in all Plasmodium species, and although variation exists in the amino acid sequence across species, the overall domain structure of a central repeat region and nonrepeat flanking regions is well conserved (see, e.g., Zhao et al. (2016) PLoS ONE 11(8): e0161607; Wahl et al. (2022) J. Exp.Med.219: e20201313, each of which is incorporated herein by reference in its entirety). CSP sequences are known (see, e.g., UniProt accession numbers A0A2L1CF52, A0A2L,1CF88, C6FGZ3, C6FH2,7 C6FHG7, M1V060, M1V0A3, M1V0B0, M1V0C4, M1V0E0, M1V9I4, M1VFN9, M1VKZ2, P02893, Q5EIJ9, Q5EIK2, Q5EIK8, Q5EIL3, Q5EIL5, Q5EIL8, Q5R2L2, Q7K740, Q8I9G5, Q8I9J3, Q8I9J4), and Table 1 includes exemplary sequences for CSP P. falciparum isolates from Asia, South America and Africa. Table 1: Exemplary Sequences for CSP P. falciparum isolates from Asia, South America and Africa Accession number Country References ), l by icAJ269945.1, de Stricker, K (Unpublished) AJ269955.1- Myanmar AJ2699601 e te a. L. m heScience.10;225(4662):593-1984), which is incorporated herein by reference in its entirety. m y y g

[0240] An exemplary CSP amino acid sequence is provided in Table 2. D. Embodiments of Malarial Sequences

[0241] An exemplary full length CSP polypeptide amino sequence from Plasmodium falciparum isolate 3D7 is presented in Table 2 as SEQ ID NO:1 and includes the following: a secretory signal (amino acids 1-18); an N- terminal domain (amino acids 19-104); a junction region (amino acids 93-104), a central domain (amino acids 105-272); and a C-terminal domain (amino acids 273-397). In exemplary SEQ ID NO:1, the N-terminal domain includes an N-terminal region (amino acids 19-80); an N-terminal end region (amino acids 81-92); and a junction region (amino acids 93-104). In exemplary SEQ ID NO:1, the junction region includes an R1 region (amino acids 93-97) and a junction (SEQ ID NO: 78) at positions 98-104. In exemplary SEQ ID NO:1, the central domainincludes a minor repeat region (amino acids 105-128) and a major repeat region (amino acids 129-272). In exemplary SEQ ID NO:1, the minor repeat region includes three repeats of the amino acid sequence NANPNVDP (SEQ ID NO:43). In exemplary SEQ ID NO:1, the major repeat region includes 35 repeats of the amino acid sequence NANP (SEQ ID NO: 98), wherein 35 repeats of the amino acid sequence NANP (SEQ ID NO: 98) are separated into two contiguous stretches, and wherein one stretch includes 17 repeats of the amino acid sequence NANP (SEQ ID NO: 98) and one includes 18 repeats of the amino acid sequence NANP (SEQ ID NO: 98) which flank an amino acid sequence of NVDP (SEQ ID NO: 97). The major repeat region includes the amino acid sequences NPNANP (SEQ ID NO:99) and NANPNA (SEQ ID NO:100). In exemplary SEQ ID NO:1, the C- terminal domain includes a C-terminal region (amino acids 273-375), a serine-valine (amino acids 376-377), and a transmembrane domain (amino acids 378-397). In exemplary SEQ ID NO:1, the C-terminal region includes a Th2R region (amino acids 314-327) and a Th3R region (amino acids 352-363). Table 2: Exemplary amino acid sequences SEQ ID Protein Sequence (Amino Acid) NO: E V P A R A TC A G T A G A C G C A AA AA A G A TA A A C U U G A G A C G U A CAAUGCAAAUCCUAAUGCAAACCCCAAUGCAAAUCCUAAUGCAAAUCCUAAUGCCAAUCC AAAUGCAAAUCCAAAUGCAAACCCAAACGCAAACCCCAAUGCAAAUCCUAAUGCCAAUC CAAAUGCAAAUCCAAAUGCAAACCCAAAUGCAAACCCAAAUGCAAACCCCAAUGCAAAU C GA U U AA U

[0242] The present disclosure, among other things, utilizes RNA technologies as a modality to express one er iant r nal ino cid g., inal 3,65, 70, 80, 90, 95, 100, 101, or 102 amino acids in length. In some embodiments, a malarial polypeptide construct described herein includes a CSP N-terminal region. In some embodiments, a malarial polypeptide construct described herein includes a portion (e.g., an antigenic portion) of a CSP N-terminal region. In some embodiments, a portion (e.g., antigenic portion) of a CSP N-terminal region is about 10, 13, 15, 20, 25, 45, 50, 55, 60, or 61 amino acids in length. In some embodiments, a malarial polypeptide construct described herein includes a CSP N-terminal end region. In some embodiments, a malarial polypeptide construct described herein includes a portion (e.g., an antigenic portion) of a CSP N-terminal end region. In some embodiments, a portion (e.g., antigenic portion) of a CSP N-terminal end region is about 8, 9, 10, or 11 amino acids in length. In some embodiments, a malarial polypeptide construct described herein includes a portion (e.g., an antigenic portion) of a CSP N-terminal start region. In some embodiments, a portion (e.g., antigenic portion) of a CSP N-terminal start region is about 8, 9, 10, or 11 amino acids in length. In some embodiments, a malarial polypeptide construct described herein includes a CSP junction region. In some embodiments, a malarial polypeptide construct described herein includes a portion (e.g., an antigenic portion) of a CSP junction region. In some embodiments, a portion (e.g., antigenic portion) of a CSP junction region is about 8, 9, 10, or 11 amino acids in length. Minor repeat region

[0245] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP minor repeat regions or portions (e.g., an antigenic portions) thereof comprising one or more repeats of an amino acid sequence of NANPNVDP (SEQ ID NO: 43).

[0246] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP polypeptide regions or portions thereof (e.g., antigenic portion) comprising one or more (e.g., 2, 3, or more) repeats of an amino acid sequence of NANPNVDP (SEQ ID NO: 43). In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP polypeptide regions or portions thereof (e.g., antigenic portion) comprising two or more repeats of an amino acid sequence of NANPNVDP (SEQ ID NO: 43). In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP polypeptide regions or portions thereof (e.g., antigenic portion) comprising exactly three repeats of an amino acid sequence of NANPNVDP (SEQ ID NO: 43).

[0247] In some embodiments, a Plasmodium CSP minor repeat region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 45 (NANPNVDPNANPNVDPNANPNVDP). In some embodiments, a Plasmodium CSP minor repeat region comprises or consists of an amino acid sequence according to SEQ ID NO: 45.

[0248] In some embodiments, the repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 43) are all contiguous with each other.

[0249] In some embodiments, a malarial polypeptide construct described herein does not comprise one or more portions of one or more Plasmodium CSP minor repeat regions (i.e., lacks or excludes a Plasmodium CSP minor repeat region or any portion thereof). C-terminal region

[0250] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP C-terminal regions (e.g., amino acids 273-375 of SEQ ID NO: 1), one or more Plasmodium CSP C-terminal region variants, or one or more portions (e.g., antigenic portions) thereof, wherein the C-terminal region does not include a transmembrane region. In some embodiments, a malarial polypeptide construct described herein includes exactly one Plasmodium CSP C-terminal region, and wherein the Plasmodium CSP C- terminal region comprises or consists of an amino acid sequence with at least 85% (e.g., at least 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acids 273-375 of SEQ ID NO: 1. In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP C-terminal region variants. In some embodiments, a Plasmodium CSP C- terminal region variant comprises or consists of an amino acid sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid sequence of SEQ ID NO: 279. In some embodiments, a malarial polypeptide construct described herein includes exactly one portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region, and wherein the portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid sequence of SEQ ID NO: 237 (PSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEK). In some embodiments, a malarial polypeptide construct described herein includes two or more portions (e.g., antigenic portions) of a Plasmodium CSP C-terminal region (e.g., amino acids 273-375 of SEQ ID NO: 1).

[0251] In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein each of the one or more portions comprises or consists of: (i) (SEQ ID NO: 57) amino acids 314-327 of SEQ ID NO: 1 (or amino acids 314-327 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions); (ii) (SEQ ID NO: 263) amino acids 311-327 of SEQ ID NO: 1 (or amino acids 311-327 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions); (iii) (SEQ ID NO: 60) amino acids 352-363 of SEQ ID NO: 1 (or amino acids 352-363 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions); (iv) (SEQ ID NO: 266) amino acids 341-364 of SEQ ID NO: 1 (or amino acids 341-364 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions); (v) (SEQ ID NO: 63) amino acids 326-374 of SEQ ID NO: 1 (or amino acids 326-374 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions), (vi) (SEQ ID NO: 66) amino acids 364-377 of SEQ ID NO: 1 (or amino acids 364-377 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions), or (vii) a combination thereof.

[0252] In some embodiments, a malarial polypeptide construct described herein includes one portion (e.g., antigenic portion) of the Plasmodium CSP C-terminal region, wherein the portion comprises or consists of: (i) amino acids 314-327 of SEQ ID NO: 1 (or amino acids 314-327 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions); (ii) amino acids 352-363 of SEQ ID NO: 1 (or amino acids 352-363 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions); (iii) amino acids 326-374 of SEQ ID NO: 1 (or amino acids 326-374 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions), (iv) amino acids 364-377 of SEQ ID NO: 1 (or amino acids 364-377 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions), or (v) a combination thereof.

[0253] In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein the one or more portions collectively comprise or consist of: (i) amino acids 314-327 of SEQ ID NO: 1 (or amino acids 314-327 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions); (ii) amino acids 352-363 of SEQ ID NO: 1 (or amino acids 352-363 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions); (iii) amino acids 326-374 of SEQ ID NO: 1 (or amino acids 326-374 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions), (iv) amino acids 364-377 of SEQ ID NO: 1 (or amino acids 364-377 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions), or (v) a combination thereof.

[0254] In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein the one or more portionscomprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region, wherein the portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 239 (YLX3X4IQX5SLST), wherein X3is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein the one or more portions comprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region, wherein the portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 240 (YLX3X4IQX5SLSTEW), wherein X3 is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein the one or more portions comprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region, wherein the portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 241 (YLX3X4IQX5SLSTEWS), wherein X3is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein the one or more portions comprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region, wherein the portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 243 (IX1X2YLX3X4IQX5SLST), wherein X1X2is EK or KE, X3is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein the one or more portions comprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region, wherein the portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 244 (IX1X2YLX3X4IQX5SLSTEW), wherein X1X2 is EK or KE, X3 is N or K, X4 is K, I, or R, and X5 is N or Y. In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein the one or more portions comprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region, wherein the portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 245 (IX1X2YLX3X4IQX5SLSTEWS), wherein X1X2is EK or KE, X3is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of a Plasmodium CSP C-terminal region, wherein the one or more portions comprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region. In some embodiments, a portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 246 (IKEYLNKIQNSLSTEWS). In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of the Plasmodium CSP C-terminal region, wherein the one or more portions comprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region. In some embodiments, a portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 237 (PSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYANDIEKKICKMEK).

[0255] In some embodiments, one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP C-terminal region variant or antigenic portion thereof. In some embodiments, a Plasmodium CSP C-terminal region variant or antigenic portion thereof comprises one or more amino acid substitutions, insertions, or deletions. In some embodiments, a Plasmodium CSP C-terminal regionvariant or antigenic portion thereof comprises one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions comprise S301N, K317E, E318Q, N321K, E357Q, A361E, or any combination thereof, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, one or more amino acid substitutions comprise S301N, K317E, E318Q, N321K, E357Q, and A361E, wherein the amino acid numbering is relative to SEQ ID NO: 1. In some embodiments, a Plasmodium CSP C-terminal region variant comprises or consists of an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 279.

[0256] In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of a Plasmodium CSP C-terminal region variant, wherein the one or more portions comprise or consist of a portion (e.g., antigenic portion) of a Plasmodium CSP C-terminal region variant. In some embodiments, a portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 277. In some embodiments, a portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 278. In some embodiments, a Plasmodium CSP C- terminal region variant comprises or consists of an amino acid sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid sequence of SEQ ID NO: 279.

[0257] In some embodiments, a malarial polypeptide construct described herein comprises a serine amino acid residue immediately following a Plasmodium CSP C-terminal region, Plasmodium CSP C-terminal region variant, or one or more portions (e.g., antigenic portions) thereof, as described herein. In some embodiments, a malarial polypeptide construct described herein comprises a serine-valine amino acid sequence immediately following a Plasmodium CSP C-terminal region, Plasmodium CSP C-terminal region variant, or one or more portions (e.g., antigenic portions) thereof, as described herein.

[0258] In some embodiments, a malarial polypeptide construct described herein does not comprise one or more portions of one or more Plasmodium CSP C-terminal regions (i.e., lacks or excludes a Plasmodium CSP C- terminal region or any portion thereof). Junction region

[0259] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP junction regions or portions thereof. In some embodiments, a junction region includes an R1 region (amino acids 93-97) and a junction (SEQ ID NO: 78) at positions 98-104.

[0260] In some embodiments, a malarial polypeptide construct described herein includes exactly one Plasmodium CSP junction region. In some embodiments, a Plasmodium CSP junction region comprises or consists of amino acids 93-104 of SEQ ID NO: 1 (or amino acids 93-104 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions). In some embodiments, a malarial polypeptide construct described herein includes one or more portions (e.g., antigenic portions) of a Plasmodium CSP junction region. In some embodiments, a portion (e.g., antigenic portion) of a Plasmodium CSP junction region comprises or consists of amino acids 93-97 of SEQ ID NO: 1. In some embodiments, a portion (e.g., an antigenic portion) of a Plasmodium CSP junction region comprises or consists of amino acids 98-104 of SEQ ID NO: 1.

[0261] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP junction regions or portions (e.g., antigenic portions) thereof, wherein the Plasmodium CSP junction region comprises or consists of an amino acid sequence that is at least 90% or 100% identical to the amino acid sequence of SEQ ID NO: 72 (KLKQPADGNPDP). In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP junction regions or portions (e.g., antigenicportions) thereof, wherein the Plasmodium CSP junction region comprises or consists of an amino acid sequence according to SEQ ID NO: 72.

[0262] In some embodiments, a malarial polypeptide construct described herein does not comprise one or more portions of one or more Plasmodium CSP junction regions (i.e., lacks or excludes a Plasmodium CSP junction region or any portion thereof). N-terminal end region

[0263] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP N-terminal end regions or portions (e.g., antigenic portions) thereof. In some embodiments, a Plasmodium CSP N-terminal end region comprises or consists of amino acids 81-92 of SEQ ID NO: 1 (or amino acids 81-92 of SEQ ID NO: 1 having 1, 2, 3, 4, or 5 amino acid substitutions). In some embodiments, a Plasmodium CSP N-terminal end region comprises or consists of an amino acid sequence that is at least 90% or at least 100% identical to the amino acid sequence of SEQ ID NO: 88 (EDNEKLRKPKHK). In some embodiments, a Plasmodium CSP N-terminal end region comprises or consists of an amino acid sequence according to SEQ ID NO: 88. In some embodiments, a malarial polypeptide construct described herein does not comprise a Plasmodium CSP N-terminal end region or any portion thereof (i.e., lacks or excludes a Plasmodium CSP N- terminal end region or any portion thereof). N-terminal region

[0264] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP N-terminal regions or portions (e.g., antigenic portions) thereof. In some embodiments, a Plasmodium CSP N-terminal region comprises or consists of amino acids 19-80 of SEQ ID NO: 1. In some embodiments, a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to amino acids 19-80 of SEQ ID NO: 1.

[0265] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise an antigenic portion of a Plasmodium CSP N-terminal region. In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of an N-terminal start region. In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the amino acid sequence of SEQ ID NO: 85.

[0266] In some embodiments, a malarial polypeptide construct described herein does not comprise a Plasmodium CSP N-terminal region or any portion thereof (i.e., lacks or excludes a Plasmodium CSP N-terminal region or any portion thereof). Major repeat region

[0267] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP major repeat regions or portions (e.g., antigenic portions) thereof. In some embodiments, a malarial polypeptide construct described herein includes exactly one Plasmodium CSP major repeat region or portion thereof, and the Plasmodium CSP major repeat region or portion thereof comprises a total of at least 2 and at most 35 repeats of the amino acid sequence NANP (SEQ ID NO: 98). In some embodiments, a portion (e.g., antigenic portion) of the Plasmodium CSP major repeat region consists of at most 18 contiguous repeats of the amino acid sequence NANP (SEQ ID NO: 98). In some embodiments, a portion (e.g., antigenic portions) of the Plasmodium CSP major repeat region consists of 18 contiguous repeats of the amino acid sequence NANP(SEQ ID NO: 98). The one or more Plasmodium CSP major repeat region or portion thereof always contains at least one repeat (e.g., one instance) of the amino acid sequence of NANP (SEQ ID NO: 98). In some embodiments, a portion (e.g., antigenic portion) of the Plasmodium CSP major repeat region comprises six (6) repeats of the amino acid sequence of NANP (SEQ ID NO: 98). In some embodiments, a portion (e.g., antigenic portion) of the Plasmodium CSP major repeat region comprises or consists of an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP. In some embodiments, a portion (e.g., antigenic portion) of the Plasmodium CSP major repeat region comprises or consists of the amino acid sequence SEQ ID NO: 101. In some embodiments, a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 101.

[0268] In some embodiments, a malarial polypeptide construct described herein does not comprise a Plasmodium CSP major repeat region or a portion of a Plasmodium CSP major repeat region comprising the amino acid sequence NPNA (SEQ ID NO: 96) (i.e., lacks or excludes a Plasmodium CSP major repeat region or a portion of a Plasmodium CSP major repeat region comprising the amino acid sequence NPNA (SEQ ID NO: 96). In some embodiments, a malarial polypeptide construct described herein does not comprise a Plasmodium CSP major repeat region or a portion of a Plasmodium CSP major repeat region comprising the amino acid sequence NANP (SEQ ID NO: 98) (i.e., lacks or excludes a Plasmodium CSP major repeat region or a portion of a Plasmodium CSP major repeat region comprising the amino acid sequence NANP (SEQ ID NO: 98). Exemplary Region Order

[0269] In some embodiments, a malarial polypeptide construct described herein optionally includes one or more of the following Plasmodium CSP polypeptide regions or portions (e.g., antigenic portions) thereof, and if present, are in the following N-terminus to C-terminus order: (i) one or more Plasmodium CSP N-terminal regions or portions (e.g., antigenic portions) thereof, for example one or more CSP N-terminal start regions or portions (e.g., antigenic portions) thereof, (ii) one or more Plasmodium CSP N-terminal end regions or portions (e.g., antigenic portions) thereof, (iii) one or more Plasmodium CSP junction regions or portions (e.g., antigenic portions) thereof, (iv) one or more repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 43), (v) one or more Plasmodium CSP major repeat regions or portions (e.g., antigenic portions) thereof, and (vi) one or more Plasmodium CSP C-terminal regions or portions (e.g., antigenic portions) thereof.

[0270] In some embodiments, a malarial polypeptide construct described herein optionally includes one or more of the following Plasmodium CSP polypeptide regions or portions (e.g., antigenic portions) thereof, and if present, are in the following N-terminus to C-terminus order: (i) one Plasmodium CSP N-terminal region or portion (e.g., antigenic portion) thereof, (ii) one Plasmodium CSP N-terminal end region or portion (e.g., antigenic portion) thereof, (iii) one Plasmodium CSP junction region or portion (e.g., antigenic portion) thereof, (iv) one or more Plasmodium CSP minor repeat sequences, (v) one Plasmodium CSP major repeat region or portion (e.g., antigenic portion) thereof, and (vi) one Plasmodium CSP C-terminal region or portion (e.g., antigenic portion) thereof. B. Secretory Signals

[0271] In some embodiments, a malarial polypeptide construct described herein includes a secretory signal, e.g., that is functional in mammalian cells. In some embodiments, a secretory signal comprises or consists of a Plasmodium secretory signal. In some embodiments, a Plasmodium secretory signal comprises or consists of a Plasmodium CSP secretory signal. In some embodiments, a Plasmodium CSP secretory signal is fromPlasmodium falciparum. In some embodiments, a Plasmodium CSP secretory signal is from Plasmodium falciparum isolate 3D7 (SEQ ID NO. 124).

[0272] In some embodiments, a utilized secretory signal is a heterologous secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal). In some embodiments, an HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal. In some embodiments, an HSV secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal according to SEQ ID NO: 251 (MGGAAARLGAVILFVVIVGLHGVRG). In some embodiments, a secretory signal comprises or consists of an Ebola virus secretory signal.

[0273] In some embodiments, a secretory signal is characterized by a length of about 15 to 30 amino acids.

[0274] In many embodiments, a secretory signal is positioned at the N-terminus of a malarial polypeptide construct described herein. In some embodiments, a secretory signal preferably allows transport of a malarial polypeptide construct with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER) or endosomal-lysosomal compartment.

[0275] In some embodiments, a secretory signal is selected from an S1S2 secretory signal (aa 1-19), an immunoglobulin secretory signal (aa 1-22), a human SPARC secretory signal, a human insulin isoform 1 secretory signal, a human albumin secretory signal, etc. Those skilled in the art will be aware of other secretory signal such as, for example, as disclosed in WO2017 / 081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOs: 1-1115 and 1728, or fragments variants thereof). In some embodiments, a malarial polypeptide construct described herein does not comprise a secretory signal.

[0276] In some embodiments, a secretory signal is one listed in Table 3, or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a signal sequence is selected from those included in the Table 3 below and / or those encoded by the sequences in Table 4 below. Table 3: Exemplary secretory signals SEQ ID NO: Signal Sequence (Amino Acid) S135 HuIgGk signal peptide METPAQLLFLLLLWLPDTTG 1 I E h h i il 1i l i MD T ILFL AAATR HSEQ ID Signal Sequence (Nucleotide) NO: C G A AG G G G149 human Ig heavy chain signal ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCAACAGGTG peptide 4 CCCACTCG TG G G CT GC G

[0277] In some embodiments, a malarial polypeptide construct described herein includes a transmembrane region (also referred to herein as a “transmembrane domain”). In some embodiments, a transmembrane region comprises or consists of a Plasmodium transmembrane region. In some embodiments, a utilized transmembrane region is one that is normally associated with CSP in nature. In some embodiments, a Plasmodium transmembrane region comprises or consists of a Plasmodium CSP glycosylphosphatidylinositol (GPI) anchor region. In some embodiments, a Plasmodium CSP GPI anchor region is from Plasmodium falciparum. In some embodiments, a Plasmodium CSP GPI anchor region is from Plasmodium falciparum isolate 3D7 (SEQ ID NO: 51), e.g., amino acids 378-397 of SEQ ID NO: 1. In some embodiments, a utilized transmembrane region is a heterologous transmembrane region.

[0278] In some embodiments, a transmembrane region is located at the N-terminus of a malarial polypeptide construct. In some embodiments, a transmembrane region is located at the C-terminus of a malarial polypeptide construct. In some embodiments, a transmembrane region is not located at the N-terminus or C- terminus of a malarial polypeptide construct.

[0279] In some embodiments, a heterologous transmembrane region does not comprise a hemagglutinin transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of an HSV transmembrane region, e.g., an HSV-1 or HSV-2 transmembrane region. In some embodiments, an HSV transmembrane region comprises or consists of an HSV gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR (SEQ ID NO: 174).

[0280] In some embodiments, a malarial polypeptide construct described herein does not comprise a transmembrane region.D. Multimerization Regions

[0281] In some embodiments, a malarial polypeptide construct described herein includes one or more multimerization regions (e.g., a heterologous multimerization region). In some embodiments, a heterologous multimerization region comprises a dimerization, trimerization or tetramerization region.

[0282] In some embodiments, a multimerization region is one described in WO2017 / 081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOs: 1116-1167, or fragments or variants thereof). Exemplary trimerization and tetramerization regions include, but are not limited to, engineered leucine zippers, fibritin foldon domain from enterobacteria phage T4, GCN4pll, GCN4-pll, and p53.

[0283] In some embodiments, a provided malarial polypeptide construct described herein is able to form a trimeric complex. For example, a provided malarial polypeptide construct may comprise a multimerization region allowing formation of a multimeric complex, such as for example a trimeric complex of a malarial polypeptide construct described herein. In some embodiments, a multimerization region allowing formation of a multimeric complex comprises a trimerization region, for example, a trimerization region described herein. In some embodiments, a malarial polypeptide construct includes a T4-fibritin-derived “foldon” trimerization region, for example, to increase its immunogenicity. In some embodiments, a malarial polypeptide construct includes a multimerization region comprising or consisting of the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPG (SEQ ID NO: 191). E. Linkers

[0284] In some embodiments, a malarial polypeptide construct described herein includes one or more linkers. In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, a linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. A linker can include any amino acid sequence and is not limited to any particular amino acids. In some embodiments, a linker comprises one or more glycine (G) amino acids. In some embodiments, a linker comprises one or more serine (S) amino acids. In some embodiments, a linker comprises a glycine-serine linker. A “glycine-serine linker” as used herein refers to a linker that comprises predominantly (e.g., 80% or more) glycine and serine amino acids. In some embodiments, a linker includes amino acids selected based on a cleavage predictor to generate highly- cleavable linkers. In some embodiments, a linker includes one or more furin cleavage sites.

[0285] In some embodiments, a linker is or comprises GGSGGGGSGG (SEQ ID NO: 193). In some embodiments, a linker is or comprises AGNRVRRSVG (SEQ ID NO: 201). In some embodiments, a linker is one presented in Table 5. In some embodiments, a linker is or comprises a sequence as set forth in WO2017 / 081082, which is incorporated herein by reference in its entirety (see SEQ ID NOs: 1509-1565, or a fragment or variant thereof).

[0286] In some embodiments, a linker is a cleavable linker. In some embodiments, a cleavable linker is or comprises AGNRVRRSVG (SEQ ID NO: 201).

[0287] In some embodiments, a malarial polypeptide construct described herein comprises a linker between a C-terminal region or portion thereof and a transmembrane region. In some embodiments, a malarial polypeptide construct described herein comprises a linker after a minor repeat sequence. In some embodiments, a malarial polypeptide construct described herein comprises a linker after a major repeat sequence or portion thereof.

[0288] Exemplary linkers are provided in the following Table 5.Table 5: Exemplary linkers SEQ ID NO: Sequence (Amino Acid)

[0289] In some embodiments, a malarial polypeptide construct described herein includes one or more self- assembling regions (e.g., a self-assembling nanoparticle region, e.g., a heterologous self-assembling nanoparticle region). In some embodiments, a self-assembling nanoparticle region is a ferritin region. In some embodiments, a ferritin region is from H. pylori. In some embodiments, a ferritin region comprises or consists of a sequence according to the amino acid sequence of DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQK AYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 248). G. Tags

[0290] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more tags may be used when designing and testing constructs (e.g., malarial polypeptide constructs described herein) in certain contexts (e.g., in vitro, ex vivo, etc.). In some embodiments, one or more tags may be directly connected to a malarial polypeptide construct through a peptide bond (e.g., at the 5’-end or 3’-end of a construct). In some embodiments, one or more tags may be connected to a malarial polypeptide construct through one or more linkers (e.g., one or more linkers described herein). In some embodiments, one or more tags may be internally embedded within a malarial polypeptide construct, wherein the one or more tags are directly connected to the construct through peptide bonds (e.g., at the 5’-end and 3’-end of the one or more tags). In some embodiments, one or more tags may be internally embedded within a malarial polypeptide construct, wherein the one or more tags are connected to the construct through one or more linkers (e.g., one or more linkers described herein).

[0291] In some embodiments, a malarial polypeptide construct described herein includes one or more tags. In some embodiments, a malarial polypeptide construct described herein includes one or more detection tags (e.g., Hibit tag, HA tag, etc.). In some embodiments, a malarial polypeptide construct described herein includes a tag that comprises or consists of a HiBit tag. In some embodiments, a HiBit tag has an amino acid sequence according to SEQ ID NO: 254.H. Embodiments of Malarial Polypeptide Constructs

[0292] In some embodiments, a malarial polypeptide construct described herein includes one or more Plasmodium CSP polypeptide regions or portions thereof as described above. Exemplary combinations of regions are described below.

[0293] In some embodiments, the malarial polypeptide construct described herein includes one or more regions or portions of CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7. 6NANP and 18 NANP CSP Constructs

[0294] In some embodiments, the malarial polypeptide construct described herein includes one or more regions or portions of CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7.

[0295] In some embodiments, the malarial polypeptide construct described herein includes at least two repeats of the amino acid sequence of NANPNVDP, two to eighteen repeats of the amino acid sequence of NANP, and a Plasmodium CSP C-terminal region or portion thereof.

[0296] As referred to herein, an “18NANP CSP Construct” includes at least: three repeats of the amino acid sequence of NANPNVDP, eighteen repeats of the amino acid sequence of NANP, and a Plasmodium CSP C- terminal region or antigenic portion thereof.

[0297] In some embodiments, the three repeats of the amino acid sequence of NANPNVDP form a Plasmodium CSP minor repeat region. In some embodiments, a Plasmodium minor repeat region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 45.

[0298] In some embodiments, the eighteen repeats of the amino acid sequence of NANP form an antigenic portion of the Plasmodium CSP major repeat region. In some embodiments, an antigenic portion of a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 101.

[0299] In some embodiments, an 18NANP CSP Construct comprises a Plasmodium CSP C-terminal region. In some embodiments, a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 54.

[0300] In some embodiments, an 18NANP CSP Construct comprises a Plasmodium CSP C-terminal region variant. In some embodiments, a Plasmodium CSP C-terminal region variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 279.

[0301] In some embodiments, an 18NANP CSP Construct comprises an antigenic portion of a Plasmodium CSP C-terminal region. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 239-246.

[0302] In some embodiments, an 18NANP CSP Construct comprises an antigenic portion of a Plasmodium CSP C-terminal region variant. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 277 or 278.

[0303] In some embodiments, an 18NANP CSP Construct comprises a serine or serine and valine immediately following the C-terminal region.

[0304] In some embodiments, an 18NANP CSP Construct comprises a Plasmodium CSP junction region. In some embodiments, a Plasmodium CSP junction region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 72.

[0305] In some embodiments, an 18NANP CSP Construct comprises a Plasmodium CSP N-terminal end region. In some embodiments, a Plasmodium CSP N-terminal end region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 88.

[0306] In some embodiments, an 18NANP CSP Construct comprises a Plasmodium CSP N-terminal region or antigenic portion thereof. In some embodiments, a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 91. In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of a Plasmodium CSP N-terminal start region. In some embodiments, a Plasmodium CSP N-terminal start region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 85.

[0307] In some embodiments, an 18NANP CSP Construct comprises one or more linkers. In some embodiments, one or more linkers are located between regions. In some embodiments, a linker is a cleavage linker. In some embodiments, a cleavage linker is positioned within an 18NANP CSP Construct between an N- terminal region or portion thereof and a C-terminal region or portion thereof.

[0308] In some embodiments, a malarial polypeptide construct can have the following structure: 18NANP CSP Construct; Secretory Signal (Sec) - 18NANP CSP Construct; 18NANP CSP Construct – Transmembrane Domain (TMD); Sec - 18NANP CSP Construct – TMD; Plasmodium falciparum (Pf) Sec - 18NANP CSP Construct; 18NANP CSP Construct – Pf TMD; Pf Sec - 18NANP CSP Construct – Pf TMD; Pf Sec - 18NANP CSP Construct – Heterologous TMD; and Pf Sec - 18NANP CSP Construct – HSV TMD.

[0309] As referred to herein, a “6NANP CSP Construct” includes at least: three repeats of the amino acid sequence of NANPNVDP, six repeats of the amino acid sequence of NANP, an asparagine-alanine positioned immediately following the six repeats of the amino acid sequence of NANP, and a Plasmodium CSP C-terminal region or antigenic fragment thereof (referred to herein as a “6NANP CSP Construct”).

[0310] In some embodiments, the three repeats of the amino acid sequence of NANPNVDP form a Plasmodium CSP minor repeat region. In some embodiments, a Plasmodium CSP minor repeat region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 45.

[0311] In some embodiments, a major repeat region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 101.

[0312] In some embodiments, a 6NANP CSP Construct comprises a Plasmodium CSP C-terminal region. In some embodiments, a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 54.

[0313] In some embodiments, an 6NANP CSP Construct comprises a Plasmodium CSP C-terminal region variant. In some embodiments, a Plasmodium CSP C-terminal region variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 279.

[0314] In some embodiments, a 6NANP CSP Construct comprises an antigenic portion of a Plasmodium CSP C-terminal region. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 239-246.

[0315] In some embodiments, an 6NANP CSP Construct comprises an antigenic portion of a Plasmodium CSP C-terminal region variant. In some embodiments, an antigenic portion of a Plasmodium CSP C-terminal region variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 277 or 278.

[0316] In some embodiments, a 6NANP CSP Construct comprises a serine or serine and valine immediately following the C-terminal region.

[0317] In some embodiments, a 6NANP CSP Construct comprises a Plasmodium CSP junction region. In some embodiments, a Plasmodium CSP junction region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 72.

[0318] In some embodiments, a 6NANP CSP Construct comprises a Plasmodium CSP N-terminal end region. In some embodiments, a Plasmodium CSP N-terminal end region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 88.

[0319] In some embodiments, a 6NANP CSP Construct comprises a Plasmodium CSP N-terminal region or antigenic portion thereof. In some embodiments, a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 91. In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of a Plasmodium CSP N-terminal start region. In some embodiments, a Plasmodium CSP N-terminal start region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 85.

[0320] In some embodiments, a 6NANP CSP Construct comprises one or more linkers. In some embodiments, one or more linkers are located between regions.

[0321] In some embodiments, a malarial polypeptide construct can have the following structure: 6NANP CSP Construct; Sec - 6NANP CSP Construct; 6NANP CSP Construct – TMD; Sec - 6NANP CSP Construct – TMD; Pf Sec - 6NANP CSP Construct; 6NANP CSP Construct – Pf TMD; Pf Sec - 6NANP CSP Construct – Pf TMD; Pf Sec - 6NANP CSP Construct – Heterologous TMD; Pf Sec - 6NANP CSP Construct – HSV TMD; Heterologous Sec - 6NANP CSP Construct – Heterologous TMD; HSV Sec - 6NANP CSP Construct – HSV TMD; Heterologous Sec - 6NANP CSP Construct – Heterologous TMD; Heterologous Sec - 6NANP CSP Construct – Multimerization Domain; or Heterologous Sec - 6NANP CSP Construct – Self-Assembly Domain. T-Cell C-Term CSP Constructs

[0322] In some embodiments, the malarial polypeptide construct described herein includes one or more regions or portions of CSP from Plasmodium falciparum, preferably from Plasmodium falciparum isolate 3D7.

[0323] As used herein, a “T-cell C-term CSP Construct” includes at least: a portion of a Plasmodium CSP C- terminal region, wherein the portion of the Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: (YLX3X4IQX5SLST), wherein X3is N or K, X4is K, I, or R, and X5is N or Y. In some embodiments, a portion of a Plasmodium CSP C-terminal region comprises an amino acid sequence according to SEQ ID NO: 239-246. In some embodiments, a portion of a Plasmodium CSP C-terminal region comprises or consists of an amino acid sequence according to SEQ ID NO: 54.

[0324] In some embodiments, a T-cell C-term CSP Construct comprises a serine or serine and valine immediately following the C-terminal region.

[0325] In some embodiments, a T-cell C-term CSP Construct further comprises a Plasmodium CSP major repeat region or an antigenic portion thereof. In some embodiments, a T-cell C-term CSP Construct further comprises a Plasmodium CSP major repeat region. In some embodiments, a Plasmodium CSP major repeat region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 103. In some embodiments, a T-cell C-term CSP Construct comprises a portion of a Plasmodium CSP major repeat region. In some embodiments, a portion of the Plasmodium CSP major repeat region comprises or consists of six repeats of the amino acid sequence of NANP. In some embodiments, an asparagine-alanine is positioned immediately following the six repeats of the amino acid sequence of NANP. In some embodiments, a portion of the Plasmodium CSP major repeat region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 101. In some embodiments, a portion of the Plasmodium CSP major repeat region comprises or consists of 18 repeats of the amino acid sequence of NANP. In some embodiments, a portion of the Plasmodium CSP major repeat region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 106.

[0326] In some embodiments, a T-cell C-term CSP Construct comprises a Plasmodium CSP minor repeat region. In some embodiments, a Plasmodium CSP minor repeat region comprises or consists of the three repeats of the amino acid sequence of NANPNVDP. In some embodiments, a Plasmodium CSP minor repeat region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 45.

[0327] In some embodiments, a T-cell C-term CSP Construct comprises a Plasmodium CSP junction region. In some embodiments, a Plasmodium CSP junction region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 72.

[0328] In some embodiments, a T-cell C-term CSP Construct comprises a Plasmodium CSP N-terminal end region. In some embodiments, a Plasmodium CSP N-terminal end region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 88.

[0329] In some embodiments, a T-cell C-term CSP Construct comprises a Plasmodium CSP N-terminal region or antigenic portion thereof. In some embodiments, a Plasmodium CSP N-terminal region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 91. In some embodiments, an antigenic portion of a Plasmodium CSP N-terminal region comprises or consists of a Plasmodium CSP N-terminal start region. In some embodiments, a Plasmodium CSP N-terminal start region comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 85.

[0330] In some embodiments, a T-cell C-term CSP Construct comprises one or more linkers. In some embodiments, one or more linkers are located between regions.

[0331] In some embodiments, a malarial polypeptide construct can have the following structure: T-cell C-term CSP Construct; Sec – T-cell C-term CSP Construct; T-cell C-term CSP Construct – TMD; Sec – T-cell C-term CSP Construct – TMD; Pf Sec – T-cell C-term CSP Construct – Heterologous TMD; Pf Sec – T-cell C-term CSP Construct – HSV TMD; Heterologous Sec - T-cell C-term CSP Construct – Heterologous TMD; HSV Sec – T-cell C-term CSP Construct – HSV TMD; Heterologous Sec - T-cell C-term CSP Construct – Multimerization Domain; HSV Sec - T-cell C-term CSP Construct – Multimerization Domain; Heterologous Sec - T-cell C-term CSP Construct – Self-Assembly Domain; or HSV Sec - T-cell C-term CSP Construct – Self-Assembly Domain. Additional Select Exemplary CSP Constructs

[0332] In some embodiments, a malarial polypeptide construct can have the following structure: N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region; Sec – N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region;N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region – TMD; ajor ajor at at nor s nor ic – nicSec – N-Terminal Region – N-Terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-Terminal Region – TMD; Pf Sec – N-Terminal Region – N-Terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-Terminal Region – Heterologous TMD; Pf Sec – N-Terminal Region – N-Terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-Terminal Region – HSV TMD; Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region; Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region; Heterologous Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region; HSV Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region; Heterologous Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Multimerization Domain; HSV Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Multimerization Domain; Heterologous Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Self-Assembly Domain; HSV Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Self-Assembly Domain; Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – TMD; Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – TMD; Heterologous Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Heterologous TMD; or HSV Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – HSV TMD. I. Exemplary Construct Sequences Table 6: Exemplary Amino Acid Sequences Encoded by RNA Constructs as Described Herein RNA Construct SEQ ID Amino Acid Sequence Y V P P V Y V P PNRNVDENANANSAVKNNNNEEPSDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQV RIKPGSANKPKDELDYANDIEKKICKMEKCSSV Y V Q V K PN N T S K PN S D T Y G A N P L Y V P A P A KI G A KI G Y IK S K PN A V A KI G175 280 MMRKLAILSVSSFLFVEALFQEYQCYGSSSNTRVLNELNYDNAGTNLYNELEMNYY GKQENWYSLKKNSRSLGENDDGNNEDNEKLRKPKHKKLKQPADGNPDPNANPNV DPNANPNVDPNANPNVDPNANPNANPNANPNANPNANPNANPNANPNANPNANP A P NS NDNA Construct SEQ ID Sequence NO: C C C G G TA A C C C G A G CT G A T A C C C G G TA A C C C G A G CT G A C C C G G TA A C G AACTCCGCCGTGAAGAACAACAACAATGAGGAACCCAGCGACAAGCACATCAAAGA GTACCTGAACAAGATCCAGAACAGCCTGAGCACCGAGTGGTCCCCATGTAGCGT GACATGCGGCAACGGAATCCAAGTGCGGATCAAGCCTGGCTCTGCCAACAAGCC A C C AG CC C T A A C G C C G G GT CT T C C AG CC C T A A T C C G C C T G C C C G G CC A A T C C C C C T C G G GCATCGTGTACTGGATGCGGCGGCACACACAGAAGGCCCCTAAGAGAATCAGACT GCCCCACATCAGATGATAA C C C G G TA A C C C T A A T G A C G T G C GT G C G C C AC G T G C GT G C G C A G C A T T T C C C C AG CC C T CTGACAAGCACATCAAAGAGTACCTGAACAAGATCCAGAACAGCCTGAGCACCGAGT GGTCCCCATGTAGCGTGACATGCGGCAACGGAATCCAAGTGCGGATCAAGCCTG GCAGCGCCAACAAGCCTAAGGACGAGCTGGACTACGCCAACGACATCGAGAAGA T G G G T G C GT G C G C T G AA C G A A A T C A AT C C A A A C G C C G G Cp y q RNA Construct SEQ ID Sequence G G G C C A A A A A A ACGAUCCCAACAGAAACGUGGACGAGAACGCAAACGCCAACAGCGCCGUGAAGAA CAACAACAAUGAGGAACCCAGCGACAAGCACAUCAAAGAGUACCUGAACAAGAU CCAGAACAGCCUGAGCACCGAGUGGUCCCCAUGUAGCGUGACAUGCGGCAACG G G C G G G C C A A A A A A A A U G G G G G G C C A A A A C A C G U G U A U C C CA CA A A G C G AC G U A94 290 AUGAUGCGGAAGCUGGCCAUCCUGAGCGUGUCCAGCUUCCUGUUUGUGGAAG CCCUGUUCCAAGAGUACCAGUGCUACGGCAGCAGCAGCAAUGGUGGAUCUGGU GGCGGAGGAUCUGGCGGAGAGGACAACGAGAAGCUGAGAAAGCCCAAGCACAA U C C CA CA G G U C G C G C G G G C C C A A A A A C G G U U A G C C G G G C C A A A A A A A A A A U U A G CGAUCCUAACGCCAAUCCUAACGUGGACCCCAACGCUAACCCCAAUGUGGACCCA AAUGCCAAUCCAAAUGUCGAUCCCAAUGCAAACCCUAACGCAAACCCGAAUGCU AACCCAAACGCUAAUCCGAACGCAAAUCCCAAUGCCAAUCCGAACGCUGGCGGA U U G A U U G G C A U A U U G A C U C C A C C A A A G U A U C C G A G AC G G CA G C A U A U U G A U G C175 282 AUGAUGCGGAAGCUGGCCAUCCUGAGCGUGUCCAGCUUCCUGUUUGUGGAAG CCCUGUUUCAGGAAUACCAGUGCUACGGAUCUUCUUCUAAUACAAGAGUGCUG AAUGAACUGAAUUACGAUAAUGCUGGAACAAAUCUCUACAAUGAACUGGAAAU U A C A U A C C A CA A AA A A U G G G CA. Exemplary Polyribonucleotides Features

[0333] Polyribonucleotides described herein encode one or more malarial polypeptide constructs described herein. In some embodiments, polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5’UTR of interest and / or a 3’ UTR of interest. In some embodiments, polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail. In some embodiments, polyribonucleotides described herein may comprise a 5’ cap, which may be incorporated during transcription, or joined to a polyribonucleotide post-transcription. 1. 5' Cap

[0334] A structural feature of mRNAs is cap structure at five-prime end (5’). Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5´ to 5´-triphosphate bridge resulting in cap0 structure (m7GpppN). In most eukaryotic mRNA and some viral mRNA, further modifications can occur at the 2'- hydroxy-group (2’-OH) (e.g., the 2'-hydroxyl group may be methylated to form 2'-O-Me) of the first and subsequent nucleotides producing “cap1” and “cap2” five-prime ends, respectively). Diamond, et al., (2014) Cytokine & growth Factor Reviews, 25:543–550, which is incorporated herein by reference in its entirety, reported that cap0-mRNA cannot be translated as efficiently as cap1-mRNA in which the role of 2'-O-Me in the penultimate position at the mRNA 5’ end is determinant. Lack of the 2'-O-met has been shown to trigger innate immunity and activate IFN response. Daffis, et al. (2010) Nature, 468:452-456; and Züst et al. (2011) Nature Immunology, 12:137-143, each of which is incorporated herein by reference in its entirety.

[0335] RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511–7526, the entire contents of each of which is hereby incorporated by reference. For example, in some embodiments, a 5’-cap structure which may be suitable in the context of the present invention is a cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation ofthe ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (“anti-reverse cap analogue”), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1 -methyl-guanosine, 2’-fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0336] The term “5'-cap” as used herein refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'-triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, a guanosine nucleoside included in a 5’ cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and / or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5’ cap comprises a 3’O methylation at a ribose (3’OMeG). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and a 3’ O methylation at a ribose (m7(3’OMeG)). It will be understood that the notation used in the above paragraph, e.g., “(m27,3’-O)G” or “m7(3’OMeG)”, applies to other structures described herein.

[0337] In some embodiments, providing an RNA with a 5'-cap disclosed herein may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase a translation efficiency and / or translation rate of an RNA, and / or increase expression of an encoded polypeptide. In some embodiments, alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.

[0338] In some embodiments, a utilized 5’ caps is a cap0, a cap1, or cap2 structure. See, e.g., Fig. 1 of Ramanathan A et al., and Fig.1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. See, e.g., Fig. 1 of Ramanathan A et al., and Fig. 1 of Decroly E et al., each of which is incorporated herein by reference in its entirety. In some embodiments, an RNA described herein comprises a cap1 structure. In some embodiments, an RNA described herein comprises a cap2.

[0339] In some embodiments, an RNA described herein comprises a cap0 structure. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G). In some embodiments, such a cap0 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as (m7)Gppp. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 2’-position of the ribose of guanosine. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 3’-position of the ribose of guanosine. In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and at the 2’-position of the ribose ((m27,2’-O)G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and at the 2’-position of the ribose ((m27,3’-O)G).

[0340] In some embodiments, a cap1 structure comprises a guanosine nucleoside methylated at the 7- position of guanine ((m7)G) and optionally methylated at the 2’ or 3’ position pf the ribose, and a 2’O methylated first nucleotide in an RNA ((m2’-O)N1). In some embodiments, a cap1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and the 3’ position of the ribose, and a 2’O methylated first nucleotide in an RNA ((m2’-O)N1). In some embodiments, a cap1 structure is connected to an RNA via a 5'- to 5'- triphosphate linkage and is also referred to herein as, e.g., ((m7)Gppp(2'-O)N1) or (m27,3’-O)Gppp(2'-O)N1), whereinN1 is as defined and described herein. In some embodiments, a cap1 structure comprises a second nucleotide, N2, which is at position 2 and is chosen from A, G, C, or U, e.g., (m7)Gppp(2'-O)N1pN2or (m27,3’-O)Gppp(2'-O)N1pN2ted es a o r cap 3 is nts, nd and ’- 1, ’ A”).or a salt there

[0346] Iure: or a salt there

[0347] In some embodiments, the 5 cap is (m2,)GpppG ( ARCA or D1 ), having a structure: or a salt there

[0348] In some embodiments, the 5’ cap is (m27,2’-O)GppSpG (“beta-S-ARCA”), having a structure: or a salt there

[0349] In some embodiments, the 5’ cap is a trinucleotide cap structure. In some embodiments, the 5’ cap is a trinucleotide cap structure comprising N1pN2, wherein N1 and N2 are as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap G*N1pN2, wherein N1and N2are as defined above and herein, and G* comprises a structure of formula (I):or a salt thereof, wherein R

[0350] In some emb.g. (m7)GpppN1pN2, (m27,2’-O)GpppN1pN2, or (m27,3’-O)GpppN1pN2), wherein N1 and N2 are as defined and described herein). In some embodiments, the 5’ cap is a trinucleotide cap1 structure (e.g., (m7)Gppp(m2’-O)N1pN2, (m27,2’-O)Gppp(m2’-O)N1pN2, (m27,3’-O)Gppp(m2’-O)N1pN2), wherein N1 and N2 are as defined and described herein. In some embodiments, the 5’ cap is a trinucleotide cap2 structure (e.g., (m7)Gppp(m2’-O)N1p(m2’-O)N2, (m27,2’-O)Gppp(m2’-O)N1p(m2’-O)N2, (m27,3’-O)Gppp(m2’-O)N1p(m2’-O)N2), wherein N1and N2are as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m27,3’-O)Gppp(m2’-O)ApG (“CleanCap AG”, “CC413”), (m27,3’-O)Gppp(m2’-O)GpG (“CleanCap GG”), (m7)Gppp(m2’-O)ApG, (m7)Gppp(m2’-O)GpG, (m27,3’-O)Gppp(m26,2’-O)ApG, and (m7)Gppp(m2’-O)ApU.

[0351] In some embodiments, the 5’ cap is (m27,3’-O)Gppp(m2’-O)ApG (“CleanCap AG”, “CC413”), having a structure: or a salt ther

[0352] In some embodiments, the 5’ cap is (m27,3’-O)Gppp(m2’-O)GpG (“CleanCap GG”), having a structure:or a salt the

[0353] , p ppp p , g or a salt ther

[0354] In some embodiments, the 5’ cap is (m7)Gppp(m2’-O)GpG, having a structure: or a salt t

[0355] In some embodiments, the 5’ cap is (m27,3’-O)Gppp(m26,2’-O)ApG, having a structure: or a salt the

[0356] , p ppp p , g or a salt thereof.

[0357] In some embodiments, the 5’ cap is a tetranucleotide cap structure. In some embodiments, the 5’ cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1, N2, and N3are as defined and described herein. In some embodiments, the 5’ cap is a tetranucleotide cap G*N1pN2pN3, wherein N1, N2, and N3 are as defined above and herein, and G* comprises a structure of formula (I): or a salt thereof, wherein R2, R

[0358] In some embodiments, the 5’ cap is a tetranucleotide cap0 structure (e.g. (m7)GpppN1pN2pN3, (m27,2’-O)GpppN1pN2pN3, or (m27,3’-O)GpppN1N2pN3), wherein N1, N2, and N3are as defined and described herein). ’2’ O 2’-areor a salt ther

[0362] In some embodiments, the 5 cap is (m )Gppp(m )Ap(m )GpG, having a structure: or a salt th2. Cap Proximal Sequences

[0363] In some embodiments, a 5’ UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5’ cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0364] In some embodiments, a cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +1 (N1) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1 and +2 (N1and N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1, +2, and +3 (N1, N2, and N3) of an RNA polynucleotide.

[0365] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and / or +5) may be included in an RNA by virtue of having been included in a cap entity (e.g., a cap1 or cap2 structure, etc.); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m27,3’-OGppp(m12’-O)ApG cap is utilized, +1 (i.e., N1) and +2 (i.e., N2) are the (m12’-O)A and G residues of the cap, and +3, +4, and +5 are added by polymerase (e.g., T7 polymerase).

[0366] In some embodiments, the 5’ cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises N1of the 5’ cap, where N1is any nucleotide, e.g., A, C, G or U. In some embodiments, the 5’ cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises N1and N2of the 5’ cap, wherein N1and N2are independently any nucleotide, e.g., A, C, G or U. In some embodiments, the 5’ cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises N1, N2, and N3of the 5’ cap, wherein N1, N2, and N3are any nucleotide, e.g., A, C, G or U.

[0367] In some embodiments, e.g., where the 5’ cap is a dinucleotide cap structure, a cap proximal sequence comprises N1 of a the 5’ cap, and N2, N3, N4 and N5, wherein N1 to N5 correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5’ cap is a trinucleotide cap structure, a cap proximal sequence comprises N1and N2of a the 5’ cap, and N3, N4and N5, wherein N1to N5correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5’ cap is a tetranucleotide cap structure, a cap proximal sequence comprises N1, N2, and N3of a the 5’ cap, and N4and N5, wherein N1to N5correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0368] In some embodiments, N1is A. In some embodiments, N1is C. In some embodiments, N1is G. In some embodiments, N1is U. In some embodiments, N2is A. In some embodiments, N2is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, N3 is A. In some embodiments, N3 is C. In some embodiments, N3is G. In some embodiments, N3is U. In some embodiments, N4is A. In some embodiments, N4is C. In some embodiments, N4is G. In some embodiments, N4is U. In some embodiments, N5is A. In some embodiments, N5 is C. In some embodiments, N5 is G. In some embodiments, N5 is U. It will be understood that each of the embodiments described above and herein (e.g., for N1through N5) may be taken singly or in combination and / or may be combined with other embodiments of variables described above and herein (e.g., 5’ caps). 3. 5’ UTR

[0369] In some embodiments, a nucleic acid (e.g., DNA, RNA) utilized in accordance with the present disclosure comprises a 5'-UTR. In some embodiments, 5’-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particularsequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element). In some embodiments a 5’ UTR comprises multiple different sequence elements.

[0370] The term “untranslated region” or “UTR” is commonly used in the art to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). As used herein, the terms “five prime untranslated region” or “5' UTR” refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide. In some embodiments, “5' UTR” refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context. In some embodiments, a 5' UTR comprises a Kozak sequence. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. In some embodiments, a 5’ UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5’ cap.

[0371] Exemplary 5’ UTRs include a human alpha globin (hAg) 5’UTR or a fragment thereof, a TEV 5’ UTR or a fragment thereof, a HSP705’ UTR or a fragment thereof, or a c-Jun 5’ UTR or a fragment thereof.

[0372] In some embodiments, an RNA disclosed herein comprises a hAg 5’ UTR or a fragment thereof.

[0373] In some embodiments, an RNA disclosed herein comprises a 5’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5’ UTR with the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 202). In some embodiments, an RNA disclosed herein comprises a 5’ UTR having the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 202). 4. PolyA Tail

[0374] In some embodiments, a polynucleotide (e.g., DNA, RNA) disclosed herein comprises a polyadenylate (polyA) sequence, e.g., as described herein. In some embodiments, a polyA sequence is situated downstream of a 3'-UTR, e.g., adjacent to a 3'-UTR.

[0375] As used herein, the term “poly(A) sequence” or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. In some embodiments, polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence. In some embodiments, polynucleotides disclosed herein comprise interrupted Poly(A) sequence. In some embodiments, RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0376] It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol.108, pp. 4009-4017, which is herein incorporated by reference).

[0377] In some embodiments, a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A)sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of" means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term “A nucleotide” or “A” refers to adenylate.

[0378] In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.

[0379] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016 / 005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference in its entirety, may be used in accordance with the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0380] In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'- end, i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.

[0381] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0382] In some embodiments, a poly A tail comprises a specific number of Adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, or about 150 or about 200. In some embodiments a poly A tail of a string construct may comprise 200 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 200 A residues. In some embodiments, a poly A tail of a string construct may comprise 180 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 180 A residues. In some embodiments, a poly A tail may comprise 150 residues or less.

[0383] In some embodiments, RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 206), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 206). In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCATATGAC. 5. 3' UTR

[0384] In some embodiments, an RNA utilized in accordance with the present disclosure comprises a 3'- UTR. As used herein, the terms “three prime untranslated region,” “3' untranslated region,” or “3' UTR” refer to a me f oIelement sequence is a 3’-UTR of amino-terminal enhancer of split (AES).

[0387] In some embodiments, an RNA disclosed herein comprises a 3’ UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3’ UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGT ATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAAC GCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAAC CCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 204). In some embodiments, an RNA disclosed herein comprises a 3’ UTR with the sequence of CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGT ATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAAC GCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAAC CCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 204).

[0388] In some embodiments, a 3’UTR is an FI element as described in WO2017 / 060314, which is herein incorporated by reference in its entirety. B. RNA Formats

[0389] At least three distinct formats useful for RNA compositions (e.g., pharmaceutical compositions) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplifying mRNA (saRNA). Each of these platforms displays unique features. In general, in all three formats, RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR),and have a polyA-tail at the 3' end. An ORF of an uRNA and modRNA vectors encode an antibody agent or portion thereof. An saRNA has multiple ORFs.

[0390] In some embodiments, the RNA described herein may have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.

[0391] The term “uracil,” as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is: .

[0392] The term “uridine,” as used herei ne of the nucleosides that can occur in RNA. The structure of uridine is:.

[0393] UTP (uridine 5’-triphosphate) hucture: .

[0394] Pseudo-UTP (pseudour ing structure:.

[0395] “Pseudouridine” iss an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0396] Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the structure:.

[0397] N1-methyl-pseudo-UTP has the o owng structure: .

[0398] Another exemplary 5U), which has the structure:.

[0399] In some embodiments, one described herein is replaced by a modifiednucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0400] In some embodiments, RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, RNA comprises a modified nucleoside in place of each uridine.

[0401] In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl- pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl- uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1-methyl- pseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and 5- methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0402] In some embodiments, the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5- oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl- pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1- taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1- deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl- pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α- thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2- thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl- 2′-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um), 3,2′-O-dimethyl- uridine (m3Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2′- F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0403] In some embodiments, the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in some embodiments, in the RNA 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. In some embodiments, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5- methyl-uridine (m5U). In some embodiments, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl- pseudouridine (m1ψ) in place of each uridine.

[0404] In some embodiments of the present disclosure, the RNA is “replicon RNA” or simply a “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA.” In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or comprises elements derived from a single-stranded (ss) RNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see José et al., Future Microbiol., 2009, vol. 4, pp.837–856, which is incorporated herein by reference in its entirety). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’-cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1–nsP4) are typically encoded together by a first ORF beginning near the 5′ terminus of the genome, while alphavirus structural proteins areencoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111–124, which is incorporated herein by reference in its entirety). Following infection, i.e., at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).

[0405] Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, a first ORF encodes an alphavirus-derived RNA-dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA. A second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding a malarial polypeptide construct described herein. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans- replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0406] Features of a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, as well as good tolerability and safety. Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, blunted immune innate immune sensor activating capacity and thus good tolerability and safety. Features of self-amplifying platform may include, for example, long duration of protein expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.

[0407] The present disclosure provides particular RNA constructs optimized, for example, for improved manufacturability, encapsulation, expression level (and / or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein. C. Codon Optimization and GC Enrichment

[0408] As used herein, the term “codon-optimized” refers to alteration of codons in a coding region of a nucleic acid molecule (e.g., a polyribonucleotide) to reflect the typical codon usage of a host organism (e.g., a subject receiving a nucleic acid molecule (e.g., a polyribonucleotide)) without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments, coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.” In some embodiments, codon-optimization may include increasing guanosine / cytosine (G / C) content of a coding region of RNA described herein as compared to the G / C content of the corresponding coding sequence of a wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.

[0409] In some embodiments, a coding sequence (also referred to as a “coding region”) is codon optimized for expression in the subject to whom a composition (e.g., a pharmaceutical composition) is to be administered (e.g., a human). Thus, in some embodiments, sequences in such a polynucleotide (e.g., a polyribonucleotide) may differ from wild type sequences encoding the relevant antigen or fragment or epitope thereof, even when the amino acid sequence of the antigen or fragment or epitope thereof is wild type.

[0410] In some embodiments, strategies for codon optimization for expression in a relevant subject (e.g., a human), and even, in some cases, for expression in a particular cell or tissue.

[0411] Various species exhibit particular bias for certain codons of a particular amino acid. Without wishing to be bound by any one theory, codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell may generally be a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are available, for example, at the "Codon Usage Database" available at www.kazusa.orjp / codon / and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.

[0412] In some embodiments, a polynucleotide (e.g., a polyribonucleotide) of the present disclosure is codon optimized, wherein the codons in the polynucleotide (e.g., the polyribonucleotide) are adapted to human codon usage (herein referred to as “human codon optimized polynucleotide”). Codons encoding the same amino acid occur at different frequencies in a subject, e.g., a human. Accordingly, in some embodiments, the coding sequence of a polynucleotide of the present disclosure is modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage, e.g., as shown in Table 9. For example, in the case of the amino acid Ala, the wild type coding sequence is preferably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with 30 a frequency of 0.10 etc. (see Table 9). Accordingly, in some embodiments, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of a polynucleotide to obtain sequences adapted to human codon usage. Table 9: Human codon usage table with frequencies indicated for each amino acid. Amino acid codon frequency Amino acid codon frequencyPhe TTT 0.43 Arg CGT 0.09 Ph TT * A 1

[04] ertan strateges or co on optmzaton an or enrc ment or uman expresson are described in WO2002 / 098443, which is incorporated by reference herein in its entirety. In some embodiments, a coding sequence may be optimized using a multiparametric optimization strategy. In some embodiments, optimization parameters may include parameters that influence protein expression, which can be, for example, impacted on a transcription level, an mRNA level, and / or a translational level. In some embodiments, exemplary optimization parameters include, but are not limited to transcription-level parameters (including, e.g., GC content, consensus splice sites, cryptic splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); mRNA-level parameters (including, e.g., RNA instability motifs, ribosomal entry sites, repetitive sequences, and combinations thereof); translation-level parameters (including,e.g., codon usage, premature poly(A) sites, ribosomal entry sites, secondary structures, and combinations thereof); or combinations thereof. In some embodiments, a coding sequence may be optimized by a GeneOptimizer algorithm as described in Fath et al. “Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression” PLoS ONE 6(3): e17596; Rabb et al., which is incorporated herein by reference in its entirety, “The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization” Systems and Synthetic Biology (2010) 4:215-225; and Graft et al. “Codon-optimized genes that enable increased heterologous expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA” Methods Mol Med (2004) 94:197-210, the entire content of each of which is incorporated herein for the purposes described herein. In some embodiments, a coding sequence may be optimized by Eurofins’ adaption and optimization algorithm “GENEius” as described in Eurofins’ Application Notes: Eurofins’ adaption and optimization software “GENEius” in comparison to other optimization algorithms, the entire content of which is incorporated by reference for the purposes described herein.

[0414] Without wishing to be bound by any particular theory, it is proposed that GC enrichment may improve translation of a payload sequence. Typically, sequences having an increased G (guanosine) / C (cytidine) content are more stable than sequences having an increased A (adenosine) / U (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by a polyribonucleotide, there are various possibilities for modification of the ribonucleic acid sequence, compared to its wild-type sequence. In particular, codons which contain A and / or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleosides. D. Embodiments of Polyribonucleotides Encoding Malarial Polypeptide Constructs

[0415] In the following, exemplary embodiments of polyribonucleotides encoding malarial polypeptide constructs are described, wherein certain terms used when describing elements thereof have the following meanings:

[0416] cap: 5'-cap structure, e.g., selected from the group consisting of m27,2'OG(5’)ppSp(5')G (in particular its D1 diastereomer), m27,3'OG(5')ppp(5')G, and m27,3'-OGppp(m12'-O)ApG.

[0417] hAg-Kozak: 5'-UTR sequence of the human alpha-globin mRNA with an optimized ʻKozak sequenceʼ to increase translational efficiency.

[0418] sec: Sequences encoding a secretory signal.

[0419] Antigen: Sequences encoding one or more malarial polypeptide constructs or portions or variants thereof (e.g., immunogenic fragments of the malarial polypeptide constructs or the immunogenic variants thereof), from Plasmodium falciparum, preferably Plasmodium falciparum isolate 3D7.

[0420] TMD: Sequences encoding a transmembrane region.

[0421] Linker: Sequences coding for peptide linkers.

[0422] FI element: The 3'-UTR is a combination of two sequence elements derived from the “amino terminal enhancer of split” (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression.

[0423] A30L70: A poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another 70 adenosine residues designed um NO: s as as ce - n ncap-hAg-Kozak-Pf Sec - 6NANP CSP Construct-FI-A30L70; cap-hAg-Kozak-6NANP CSP Construct – Pf TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec - 6NANP CSP Construct – Pf TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec - 6NANP CSP Construct – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec - 6NANP CSP Construct – HSV TMD-FI-A30L70; cap-hAg-Kozak-Heterologous Sec - 6NANP CSP Construct – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-HSV Sec - 6NANP CSP Construct – HSV TMD-FI-A30L70; cap-hAg-Kozak-Heterologous Sec - 6NANP CSP Construct – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-Heterologous Sec - 6NANP CSP Construct – Multimerization Domain-FI-A30L70; cap-hAg-Kozak-Heterologous Sec - 6NANP CSP Construct – Self-Assembly Domain-FI-A30L70; cap-hAg-Kozak-T-cell C-term CSP Construct-FI-A30L70; cap-hAg-Kozak-Sec – T-cell C-term CSP Construct-FI-A30L70; cap-hAg-Kozak-T-cell C-term CSP Construct – TMD-FI-A30L70; cap-hAg-Kozak-Sec – T-cell C-term CSP Construct – TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – T-cell C-term CSP Construct – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – T-cell C-term CSP Construct – HSV TMD-FI-A30L70; cap-hAg-Kozak-Heterologous Sec - T-cell C-term CSP Construct – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-HSV Sec – T-cell C-term CSP Construct – HSV TMD-FI-A30L70; cap-hAg-Kozak-Heterologous Sec - T-cell C-term CSP Construct – Multimerization Domain-FI- A30L70; cap-hAg-Kozak-HSV Sec - T-cell C-term CSP Construct – Multimerization Domain-FI-A30L70; cap-hAg-Kozak-Heterologous Sec - T-cell C-term CSP Construct – Self-Assembly Domain-FI-A30L70; cap-hAg-Kozak-HSV Sec - T-cell C-term CSP Construct – Self-Assembly Domain-FI-A30L70; cap-hAg-Kozak-N-Term Start CSP Construct-FI-A30L70; cap-hAg-Kozak-Sec – N-Term Start CSP Construct-FI-A30L70; cap-hAg-Kozak-N-Term Start CSP Construct– TMD-FI-A30L70; cap-hAg-Kozak-Sec – N-Term Start CSP Construct – TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – T-cell C-term CSP Construct – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – T-cell C-term CSP Construct – HSV TMD-FI-A30L70; cap-hAg-Kozak-N-Terminal Region CSP Construct-FI-A30L70; cap-hAg-Kozak-Sec – N-Terminal Region CSP Construct-FI-A30L70; cap-hAg-Kozak-N-Terminal Region CSP Construct – TMD-FI-A30L70; cap-hAg-Kozak-Sec – N-Terminal Region CSP Construct – TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – N-Terminal Region CSP Construct – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – N-Terminal Region CSP Construct – HSV TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – Tag – N-Terminal Region CSP Construct – HSV TMD-FI-A30L70; cap-hAg-Kozak-N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region-FI-A30L70; cap-hAg-Kozak-Sec – N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region-FI-A30L70; cap-hAg-Kozak-N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region - TMD-FI-A30L70;cap-hAg-Kozak-Sec – N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region - TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region-FI-A30L70; cap-hAg-Kozak-Pf Sec – N-terminal Region – N-terminal End Region – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – C-terminal Region – Pf TMD-FI- A30L70; cap-hAg-Kozak-N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – C-Terminal Region-FI-A30L70; cap-hAg-Kozak-N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – Antigenic Portion of a C- Terminal Region-FI-A30L70; cap-hAg-Kozak-Sec – N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – C-Terminal Region-FI- A30L70; cap-hAg-Kozak-Sec – N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – Antigenic Portion of a C- Terminal Region C-Terminal Region-FI-A30L70; cap-hAg-Kozak-N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – C-Terminal Region – TMD-FI- A30L70; cap-hAg-Kozak-N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – Antigenic Portion of a C- Terminal Region – TMD-FI-A30L70; cap-hAg-Kozak-Sec – N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – C-Terminal Region – TMD- FI-A30L70; cap-hAg-Kozak-Sec – N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – Antigenic Portion of a C- Terminal Region – TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – C-Terminal Region – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – Antigenic Portion of a C-Terminal Region – Heterologous TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – C-Terminal Region – HSV TMD-FI-A30L70; cap-hAg-Kozak-Pf Sec – N-Terminal Start Region – Linker – N-terminal End Region – Junction Region – Minor Repeat Region - Antigenic Portion of a Major Repeat Region – Antigenic Portion of a C-Terminal Region – HSV TMD-FI-A30L70;cap-hAg-Kozak-N-Terminal Region – N-Terminal End Region – Cleavable Linker – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of a C- of n – of ion of ion of eat of a r of acap-hAg-Kozak-HSV Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Multimerization Domain-FI-A30L70; cap-hAg-Kozak-Heterologous Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Self-Assembly Domain-FI-A30L70; cap-hAg-Kozak-HSV Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Self-Assembly Domain-FI-A30L70; cap-hAg-Kozak-Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – TMD-FI-A30L70; cap-hAg-Kozak-Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – TMD-FI-A30L70; cap-hAg-Kozak-Heterologous Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – Heterologous TMD-FI-A30L70; or cap-hAg-Kozak-HSV Sec – Junction Region – Minor Repeat Region – Antigenic Portion of a Major Repeat Region – Antigenic Portion of C-Terminal Region – HSV TMD-FI-A30L70.

[0428] In some embodiments, the different elements (sec, Antigen, TMD) may be linked by one or more linkers, e.g., a linker selected from an amino acid sequence as defined in Table 5. In some embodiments, a linker has the amino acid sequence GGSGGGGSGG. In some embodiments, a linker has the amino acid sequence GGGS. In some embodiments, a linker has the amino acid sequence GGGGSGGGGSGGGGS. In some embodiments, a linker has the amino acid sequence AGNRVRRSVG.

[0429] In some embodiments, the sequence encoding a malarial polypeptide construct described herein comprises a modified nucleoside replacing (partially or completely, preferably completely) uridine, wherein the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine.

[0430] In some embodiments, the sequence encoding a malarial polypeptide construct described herein is codon-optimized.

[0431] In some embodiments, the G / C content of the sequence encoding a malarial polypeptide construct described herein is increased compared to the wild type coding sequence.

[0432] In some embodiments, the RNA (in particular, mRNA) described herein comprises: a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 203, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 203; a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 205, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 205; and a poly-A sequence comprising the nucleotide sequence of SEQ ID NO: 207.

[0433] In some embodiments, the RNA (in particular, mRNA) described herein comprises: m27,3’-OGppp(m12’-O) ApG as capping structure at the 5'-end of the mRNA; a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 203, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 203;a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 205, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 205; and a poly-A sequence comprising the nucleotide sequence of SEQ ID NO: 207.

[0434] In some embodiments, the RNA is unmodified. In some embodiments, the RNA is modified. In some embodiments, the RNA comprises N1-methyl-pseudouridine (m1ψ) in place of at least one uridine (e.g., in place of each uridine).

[0435] In some embodiments, the RNA (in particular, mRNA) described herein comprises: m27,3’-OGppp(m12’-O) ApG as capping structure at the 5'-end of the mRNA; a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 203, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 203; a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 205, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 205; and a poly-A sequence comprising the nucleotide sequence of SEQ ID NO: 207; and N1-methyl-pseudouridine (m1ψ) in place of at least one uridine (e.g., in place of each uridine).

[0436] In some embodiments, a malarial polypeptide construct described herein includes one or more malarial polypeptides or portions thereof from Plasmodium falciparum.

[0437] In some embodiments, a malarial polypeptide construct described herein includes one or more regions or portions thereof derived from a Plasmodium falciparum CSP protein, an immunogenic variant thereof, or an immunogenic fragment of the Plasmodium falciparum CSP protein or the immunogenic variant thereof. Thus, in some embodiments, the RNA, e.g., mRNA, used in the present disclosure encodes an amino acid sequence comprising an Plasmodium falciparum CSP protein, an immunogenic variant thereof, or an immunogenic fragment of the Plasmodium falciparum CSP protein or the immunogenic variant thereof.

[0438] In some embodiments, RNA (in particular, mRNA) described herein (e.g., contained in the compositions / formulations of the present disclosure and / or used in the methods of the present disclosure) may be presented as a product containing the vaccine RNA as active substance and other ingredients comprising: ALC-0315 ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol.

[0439] In some embodiments, the RNA (in particular, mRNA) described herein is formulated or is to be formulated as a liquid, a solid, or a combination thereof.

[0440] In some embodiments, the RNA (in particular, mRNA) described herein is formulated or is to be formulated for injection.

[0441] In some embodiments, the RNA (in particular, mRNA) described herein is formulated or is to be formulated for intramuscular administration.

[0442] In some embodiments, the RNA (in particular, mRNA) described herein is formulated or is to be formulated as a composition, e.g., a pharmaceutical composition.

[0443] In some embodiments, the composition comprises a cationically ionizable lipid.

[0444] In some embodiments, the composition comprises a cationically ionizable lipid and one or more additional lipids. In some embodiments, the one or more additional lipids are selected from polymer-conjugated lipids, neutral lipids, and combinations thereof. In some embodiments, the neutral lipids include phospholipids,steroid lipids, and combinations thereof. In some embodiments, the one or more additional lipids are a combination of a polymer-conjugated lipid, a phospholipid, and a steroid lipid.

[0445] In some embodiments, the composition comprises a cationically ionizable lipid; a polymer- conjugated lipid which is a PEG-conjugated lipid; cholesterol; and a phospholipid. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE.

[0446] In some embodiments, the composition comprises a cationically ionizable lipid; a polymer- conjugated lipid which is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; cholesterol; and a phospholipid. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE.

[0447] In some embodiments, the composition comprises a cationically ionizable lipid which is ((4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); a polymer-conjugated lipid which is 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; cholesterol; and a phospholipid. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE. In some embodiments, at least a portion of (i) the RNA, (ii) the cationically ionizable lipid, and if present, (iii) the one or more additional lipids is present in particles. In some embodiments, the particles are nanoparticles, such as lipid nanoparticles (LNPs).

[0448] In some embodiments, the composition, in particular the pharmaceutical composition, is a vaccine.

[0449] In some embodiments, the composition, in particular the pharmaceutical composition, further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0450] In some embodiments, the RNA and / or the composition, in particular the pharmaceutical composition, is / are a component of a kit.

[0451] In some embodiments, the kit further comprises instructions for use of the RNA for inducing an immune response against Plasmodium falciparum in a subject.

[0452] In some embodiments, the kit further comprises instructions for use of the RNA for therapeutically or prophylactically treating a Plasmodium falciparum infection in a subject.

[0453] In some embodiments, the subject is a human.

[0454] In some embodiments, the RNA (in particular, mRNA), e.g., RNA encoding a malarial polypeptide construct, described in the present disclosure is non-immunogenic. RNA encoding an immunostimulant may be administered according to the present disclosure to provide an adjuvant effect. The RNA encoding an immunostimulant may be standard RNA or non-immunogenic RNA.

[0455] As used herein, an “ERMA” construct is an “RNA construct,” and, for example, “ERMA 1” corresponds to “RNA Construct 1”, “ERMA 2” corresponds to “RNA construct 2,” etc. IV. RNA Delivery Technologies

[0456] Provided polyribonucleotides may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNAs, or delivery mediated by viral and / or non-viral vectors, polymer-based vectors, lipid compositions, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and / or polypeptide-based vectors. See, e.g., Wadhwa et al. “Opportunities and Challenges in the Delivery of mRNA-Based Vaccines” Pharmaceutics (2020) 102 (27 pages), the content of which is incorporated herein by reference, for information on various approaches that may be useful for delivery polyribonucleotides described herein.

[0457] In some embodiments, one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery (e.g., administration).

[0458] In some embodiments, lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and / or engineered for systemic delivery of the RNA to target cells (e.g., liver cells). In someembodiments, such lipid nanoparticles may be particularly useful to deliver polyribonucleotides when polyribonucleotides are intravenously or intramuscularly administered to a subject. A. Lipid Compositions 1. Lipids and Lipid-Like Materials

[0459] The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and / or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

[0460] Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0461] A "lipid-like material" is a substance that is structurally and / or functionally related to a lipid but may not be considered a lipid in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.

[0462] Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.

[0463] Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterols and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol- containing metabolites such as cholesterol.

[0464] Fatty acids are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain tobend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.

[0465] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.

[0466] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

[0467] Sphingolipids are members of a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0468] Sterols, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with the glycerophospholipids and sphingomyelins.

[0469] Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty- acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0470] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0471] Lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

[0472] In some embodiments, suitable lipids or lipid-like materials for use in the present disclosure include those described in WO2020 / 128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.2. Cationic or Cationically Ionizable Lipids or Lipid-Like Materials

[0473] In some embodiments cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In one embodiment, cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0474] Cationic lipids or lipid-like materials are characterized in that they have a net positive charge (e.g., at a relevant pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

[0475] In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

[0476] In some embodiments, a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.

[0477] Examples of cationic lipids include, but are not limited to 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3- dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2- dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en- 3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′- oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3- Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K- XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9- tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium- propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1- aminium bromide (DMORIE), di((Z)-non-2-en-1-yl) 8,8'- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan- 1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), Di((Z)-non-2-en-1-yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2- dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}- ethylamino)propionamide (lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1 ,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1 -(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing. Further suitable cationic lipids for use in the present disclosure include those described in WO2020 / 128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein. Further suitable cationic lipids for use in the present disclosure include those described in WO2010 / 053572 (including Cl 2-200 described at paragraph

[0225] ) and WO2012 / 170930, both of which are incorporated herein by reference for the purposes described herein. Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1, which is incorporated herein by reference in its entirety).

[0478] In some embodiments, formulations that are useful for pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) compositions as described herein can comprise at least one cationic lipid. Representative cationic lipids include, but are not limited to, 1 ,2-dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3- dimethylaminopropane (DLinDAP), 1 ,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2- linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1 ,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-1 ,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1 ,2-propanediol (DOAP), 1 ,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2- dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1 ,3]- dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120, which is incorporated herein by reference in its entirety).

[0479] In some embodiments, amino or cationic lipids useful in accordance with the present disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant speciesand does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise be suitable in the context of the present invention.

[0480] In some embodiments, a protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

[0481] In some embodiments, a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of total lipid present in a lipid composition utilized in accordance with the present disclosure. 3. Additional Lipids or Lipid-Like Materials

[0482] In some embodiments, formulations utilized in accordance with the present disclosure may comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid- like materials. In some embodiments, optimizing a formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable / cationic lipid or lipid-like material may, for example, enhance particle stability and efficacy of nucleic acid delivery.

[0483] In some embodiments, a lipid or lipid-like material may be incorporated which may or may not affect the overall charge of particles. In certain embodiments, such lipid or lipid-like material is a non-cationic lipid or lipid-like material.

[0484] In some embodiments, a non-cationic lipid may comprise, e.g., one or more anionic lipids and / or neutral lipids. An "anionic lipid" is negatively charged (e.g., at a selected pH).

[0485] A "neutral lipid" exists either in an uncharged or neutral zwitterionic form (e.g., at a selected pH). In some embodiments, a formulation comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0486] Specific exemplary phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl- phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl- phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.

[0487] In certain embodiments, a formulation utilized in accordance with the present disclosure includes DSPC or DSPC and cholesterol.

[0488] In certain embodiments, formulations utilized in accordance with the present disclosure include both a cationic lipid and an additional (non-cationic) lipid.

[0489] In some embodiments, formulations herein include a polymer conjugated lipid such as a pegylated lipid. "Pegylated lipids" comprise both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

[0490] Without wishing to be bound by theory, the amount of (total) cationic lipid compared to the amount of other lipid(s) in formulation may affect important characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

[0491] In some embodiments, a non-cationic lipid, in particular a neutral lipid, (e.g., one or more phospholipids and / or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in a formulation. 4. Lipoplex Particles

[0492] In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipoplex particles.

[0493] An "RNA lipoplex particle" contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, an RNA lipoplex particle is a nanoparticle.

[0494] In certain embodiments, RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0495] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0496] In some embodiments, RNA lipoplex particles have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameterthat ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0497] RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration. The RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM. Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplex particles comprise 1,2-di-O- octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE).

[0498] Spleen targeting RNA lipoplex particles are described in WO 2013 / 143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression in the lung and / or liver occurs. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and / or macrophages. 5. Lipid Nanoparticles (LNPs)

[0499] In some embodiments, nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs). In some embodiments, LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0500] In some embodiments, an LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0501] In some embodiments, an LNP comprises a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid; and an RNA, encapsulated within or associated with the lipid nanoparticle.

[0502] In some embodiments, a neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

[0503] In some embodiments, a sterol is cholesterol.

[0504] In some embodiments, a polymer conjugated lipid is a pegylated lipid. In some embodiments, a pegylated lipid has the following structure: or a pharmaceutically accepta rein: R12and R13are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some embodiments, R12and R13are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, w has a mean value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R12and R13are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.

[0505] In some embodiments, a pegylated lipid is DMG-PEG 2000, e.g., having the following structure: .

[0506] ula (III):R3G3R2or a pharmaceutically acceptable salt, t mer thereof, wherein: 1one of L or L2is –O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, - C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1or L2is –O(C=O)-, -(C=O)O-, - C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or - NRaC(=O)O- or a direct bond; G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3is C1-C24alkylene, C1-C24alkenylene, C3-C8cycloalkylene, C3-C8cycloalkenylene; Rais H or C1-C12 alkyl; R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl; R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or –NR5C(=O)R4; R4is C1-C12 alkyl; R5is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0507] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB): wherein: A is a 3 toR6is, at each occurrence, independently H, OH or C1-C24alkyl; n is an integer ranging from 1 to 15.

[0508] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0509] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID): wherein y and z are e

[0510] In any of the foregoing embodiments of Formula (III), one of L or L is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some different embodiments of any of the foregoing, L1and L2are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0511] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF): .

[0512] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):; .

[0513] g g , g g g m 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0514] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0515] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24 alkyl. In other embodiments, R6is OH.

[0516] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24 alkenylene.

[0517] In some other foregoing embodiments of Formula (III), R1or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure: , wherein:R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0518] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7ais H. For example, in some embodiments, R7ais H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7bis C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n- propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0519] In different embodiments of Formula (III), R1or R2, or both, has one of the following structures:; ; ; ; )R4or –

[0521] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in in Table 10 below. Table 10: Exemplary Compounds of Formula (III). No.StructureNo.StructureNo.StructureNo.StructureNo.StructureNo.Structure

[0522] In variset forth in Table 11 below. Table 11: Exemplary Cationic Lipid Structures No. StructureNo. Structure O O

[0523] In s id-like material(lipidoid). In some embodiments, a cationic lipid has the following structure: .

[0524] eter) of about30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 60 nm to about 120 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0525] In some embodiments, lipid nanoparticles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, lipid nanoparticles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3. The “polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so- called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken a...

Claims

CLAIMS 1. A polyribonucleotide encoding a polypeptide that comprises one or more Plasmodium CSP polypeptide regions or antigenic portions thereof.

2. The polyribonucleotide of claim 1, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise: (i) at least two repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 43), (ii) two to eighteen repeats of the amino acid sequence of NANP (SEQ ID NO: 98), and (iii) a Plasmodium CSP C-terminal region, a Plasmodium CSP C-terminal region variant, or an antigenic portion thereof.

3. The polyribonucleotide of claim 1 or 2, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise, in N-terminal to C-terminal order: (i) the at least two repeats of the amino acid sequence of NANPNVDP (SEQ ID NO: 43), (ii) the two to eighteen repeats of the amino acid sequence of NANP (SEQ ID NO: 98), and (iii) the Plasmodium CSP C-terminal region, a Plasmodium CSP C-terminal region variant, or an antigenic portion thereof.

4. The polyribonucleotide of any one of claims 1-3, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise an antigenic portion of a Plasmodium CSP C-terminal region or Plasmodium CSP C-terminal region variant.

5. The polyribonucleotide of any one of claims 1-4, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP C-terminal region.

6. The polyribonucleotide of any one of claims 1-5, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP C-terminal region variant or antigenic portion thereof.

7. The polyribonucleotide of any one of claims 2-6, wherein the polypeptide comprises a serine immediately following the Plasmodium CSP C-terminal region, Plasmodium CSP C-terminal region variant, or antigenic portion thereof.

8. The polyribonucleotide of any one of claims 2-6, wherein the polypeptide comprises a serine-valine sequence immediately following the Plasmodium CSP C-terminal region, Plasmodium CSP C-terminal region variant, or antigenic portion thereof.

9. The polyribonucleotide of any one of claims 1-8, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP major repeat region or antigenic portion thereof.

10. The polyribonucleotide of any one of claims 1-9, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP minor repeat region or antigenic portion thereof.

11. The polyribonucleotide of any one of claims 1-10, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP N-terminal region or antigenic portion thereof.

12. The polyribonucleotide of any one of claims 1-11, wherein the one or more Plasmodium CSP polypeptide regions or antigenic portions thereof comprise a Plasmodium CSP junctional region.

13. The polyribonucleotide of any one of claims 1-12, wherein the polypeptide comprises one or more linkers.

14. The polyribonucleotide of claim 13, wherein the one or more linkers are located: (i) between two Plasmodium CSP polypeptide regions or antigenic portions thereof; (ii) between a Plasmodium CSP polypeptide region or antigenic portion thereof and a transmembrane region; (iii) between a Plasmodium CSP polypeptide region or antigenic portion thereof and a self-assembling region; and / or (iv) between a Plasmodium CSP polypeptide region or antigenic portion thereof and a multimerization region.

15. The polyribonucleotide of any one of claims 1-14, wherein the polypeptide comprises a secretory signal.

16. The polyribonucleotide of any one of claims 1-15, wherein the polypeptide comprises a transmembrane region.

17. The polyribonucleotide of any one of claims 1-14, wherein the polypeptide comprises a multimerization region.

18. The polyribonucleotide of any one of claims 1-17, wherein the polypeptide comprises a self-assembling region.

19. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junctional region, (v) a Plasmodium CSP minor repeat region, (vi) an antigenic portion of a Plasmodium CSP major repeat region, and (vii) a Plasmodium CSP C-terminal region.

20. The polyribonucleotide of claim 19, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

7.

21. The polyribonucleotide of any one of claims 19-20, wherein the polyribonucleotide comprises or consists of a ribonucleic acid sequence with at least 85% sequence identity to the ribonucleic acid sequence according to SEQ ID NO:

9.

22. The polyribonucleotide of claim 20, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

10.

23. The polyribonucleotide of claim 20 or claim 22, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the ribonucleic acid sequence according to SEQ ID NO:

12.

24. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junctional region, (v) a Plasmodium CSP minor repeat region, (vi) an antigenic portion of a Plasmodium CSP major repeat region, (vii) a Plasmodium CSP C-terminal region, and (viii) a transmembrane region.

25. The polyribonucleotide of claim 24, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

4.

26. The polyribonucleotide of any one of claims 24-25, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

6.

27. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a first linker, (v) a Plasmodium CSP junctional region, (vi) a Plasmodium CSP minor repeat region, (vii) an antigenic portion of a Plasmodium CSP major repeat region, (viii) a Plasmodium CSP C-terminal region, (ix) a second linker, and (x) a transmembrane region.

28. The polyribonucleotide of claim 27, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

25.

29. The polyribonucleotide of any one of claims 27-28, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

27.

30. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junctional region, (v) a Plasmodium CSP minor repeat region, (vi) a Plasmodium CSP major repeat region, (vii) a linker, and (viii) a transmembrane region.

31. The polyribonucleotide of claim 30, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

28.

32. The polyribonucleotide of any one of claims 30-31, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

30.

33. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP junctional region, (iii) a Plasmodium CSP minor repeat region, (iv) an antigenic portion of a Plasmodium CSP major repeat region, (v) a first linker, (vi) an antigenic portion of a Plasmodium CSP C-terminal region, (vii) a serine-valine sequence, (viii) a second linker, and (ix) a multimerization region.

34. The polyribonucleotide of claim 33, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

31.

35. The polyribonucleotide of any one of claims 33-34, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO: 33.

36. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP junctional region, (iii) a Plasmodium CSP minor repeat region, (iv) an antigenic portion of a Plasmodium CSP major repeat region, (v) a first linker, (vi) an antigenic portion of a Plasmodium CSP C-terminal region, (vii) a serine-valine sequence, (viii) a second linker, and (ix) a self-assembling region.

37. The polyribonucleotide of claim 36, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

34.

38. The polyribonucleotide of any one of claims 36-37, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

36.

39. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP junctional region, (iii) a Plasmodium CSP minor repeat region, (iv) an antigenic portion of a Plasmodium CSP major repeat region, (v) a first linker, (vi) an antigenic portion of a Plasmodium CSP C-terminal region, (vii) a serine-valine sequence, (viii) a second linker, and (ix) a transmembrane region.

40. The polyribonucleotide of claim 39, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

40.

41. The polyribonucleotide of any one of claims 39-40, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

42.

42. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) a Plasmodium CSP N-terminal region, (iii) a Plasmodium CSP N-terminal end region, (iv) a Plasmodium CSP junctional region,(v) a Plasmodium CSP minor repeat region, (vi) a Plasmodium CSP major repeat region, (vii) a Plasmodium CSP C-terminal region variant, and (viii) a transmembrane region.

43. The polyribonucleotide of 42, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

280.

44. The polyribonucleotide of any one of claims 42-43, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

282.

45. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) an antigenic portion of a Plasmodium CSP N-terminal region, (iii) a first linker, (iv) a Plasmodium CSP N-terminal end region, (v) a Plasmodium CSP junctional region, (vi) a Plasmodium CSP minor repeat region, (vii) an antigenic portion of a Plasmodium CSP major repeat region, (viii) a Plasmodium CSP C-terminal region, (ix) a serine-valine sequence, (x) a second linker, and (xi) a transmembrane region.

46. The polyribonucleotide of 45, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

285.

47. The polyribonucleotide of any one of claims 45-46, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

287.

48. The polyribonucleotide of claim 1, wherein the polypeptide comprises: (i) a secretory signal, (ii) an antigenic portion of a Plasmodium CSP N-terminal region, (iii) a first linker, (iv) a Plasmodium CSP N-terminal end region, (v) a Plasmodium CSP junctional region, (vi) a Plasmodium CSP minor repeat region, (vii) an antigenic portion of a Plasmodium CSP major repeat region, (viii) a second linker, (ix) an antigenic portion of a Plasmodium CSP C-terminal region,(x) a serine-valine sequence, (xi) a third linker, and (xii) a transmembrane region.

49. The polyribonucleotide of claim 48, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

288.

50. The polyribonucleotide of any one of claims 48-49, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

290.

51. The polyribonucleotide of any one of claims 48-49, wherein the polypeptide comprises or consists of an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to SEQ ID NO:

291.

52. The polyribonucleotide of any one of claims 48-49 or claim 51, wherein the polyribonucleotide comprises or consists of a nucleic acid sequence with at least 85% sequence identity to the nucleic acid sequence according to SEQ ID NO:

293.

53. An RNA construct comprising in 5' to 3' order: (i) a 5' UTR that comprises or consists of a modified human alpha-globin 5'-UTR; (ii) a polyribonucleotide of any one of claims 1-50; (iii) a 3' UTR that comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA; and (iv) a polyA tail sequence.

54. A composition comprising one or more polyribonucleotides of any one of claim 1-52.

55. A composition comprising one or more RNA constructs of claim 53.

56. The composition of claim 54 or 55, wherein the composition further comprises lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes, wherein the one or more polyribonucleotides are fully or partially encapsulated within the lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), or liposomes.

57. The composition of claim 56, wherein the lipid nanoparticles each comprise: (a) a polymer-conjugated lipid; (b) a cationic lipid; and (c) one or more neutral lipids.

58. A pharmaceutical composition comprising the composition of any one of claims 54-58 and at least one pharmaceutically acceptable excipient.

59. A method comprising administering a polyribonucleotide of any one of claims 1-52 to a subject.

60. A method comprising administering an RNA construct of claim 53 to a subject.

61. A method comprising administering a composition of any one of claims 54-57 to a subject.

62. A method comprising administering one or more doses of the pharmaceutical composition of claim 58 to a subject.

63. The pharmaceutical composition of claim 58 for use in the treatment of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject.

64. The pharmaceutical composition of claim 58 for use in the prevention of a malaria infection comprising administering one or more doses of the pharmaceutical composition to a subject.

65. A combination comprising: (i) a first pharmaceutical composition comprising a first polyribonucleotide, wherein the first polyribonucleotide is a polyribonucleotide of any one of claims 1-52; and (ii) a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide encodes a second polypeptide, and the second polypeptide comprises one or more Plasmodium T-cell antigens.

66. A method comprising administering a combination of claim 65 to a subject.

67. Use of the pharmaceutical composition of claim 58 in the treatment of a malaria infection.

68. Use of the pharmaceutical composition of claim 58 in the prevention of a malaria infection.

69. Use of the pharmaceutical composition of claim 58 in inducing an anti-malaria immune response in a subject.

70. A host cell comprising a polyribonucleotide of any one of claims 1-52.

71. A host cell comprising an RNA construct of claim 53.

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